Treatment methods for disease using co-localized cells and Sertoli cells obtained from a cell line

ABSTRACT

A method of treating a disease is provided that results from a deficiency of a biological factor which comprises administering to a mammal Sertoli cells and cells that produce the biological factor. A method of treating diabetes mellitus is carried out by transplanting pancreatic islet of Langerhans cells in conjunction with Sertoli cells to create an immunologically privileged site. A method of creating an immunologically privileged site and providing cell stimulatory factors in a mammal for transplants is also carried out. A method of co-localizing islet cells with Sertoli cells and the use of the co-localized product for treating diabetes mellitus is further provided. Further described is a method of creating systemic tolerance to foreign antigens. A method of enhancing the viability, maturation, proliferation of functional capacity of cells in tissue culture is also provided. In addition, a pharmaceutical composition comprising Sertoli cells and cells that produce a biological factor is provided.

This invention was made with United States government support undergrant DK42421 awarded by the National Institutes of Health. The UnitedStates Government may have certain rights in the invention.

CROSS-REFERENCE OF RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 08/485,340filed on Jun. 7, 1995, now U.S. Pat. No. 5,849,285, which is acontinuation-in-part of U.S. Ser. No. 08/421,641 filed on Apr. 13, 1995,now U.S. Pat. No. 5,725,854 which is a continuation-in-part of U.S. Ser.No. 08/211,695 filed on Apr. 13, 1994 now abandoned.

FIELD OF THE INVENTION

Transplants of healthy organs or cells into a patient suffering from adisease are often rejected by the body due to an immune responseinitiated in response to the foreign tissue or cells. The presentinvention provides a method of cellular transplantation in which animmunologically privileged site is created and cell stimulatory factorsare produced, thus alleviating the rejection associated withconventional transplantation therapy. Specifically, the presentinvention describes a method of treating a disease that results from adeficiency of a biological factor which comprises administering to amammal Sertoli cells and cells that produce the biological factor. Inparticular, the present invention describes a method of treatingdiabetes mellitus by transplanting pancreatic islet of Langerhans cellsin conjunction with Sertoli cells to create an immunologicallyprivileged site and to provide pancreatic islet cell stimulatoryfactors. A method of creating an immunologically privileged site andproviding cell stimulatory factors in a mammal for transplants isfurther described by the present invention. A method of creatingsystemic tolerance to transplants is further provided by the presentinvention. The present invention further describes a method of enhancingthe maturation, proliferation and functional capacity of cells in tissueculture by coculturing these cells with Sertoli cells. A method ofenhancing the recovery rate and viability of frozen cells, and inparticular factor producing cells, in tissue culture by co-culturingthese cells with Sertoli cells is also described herein. Another aspectof the present invention is directed to a method of co-localizingSertoli cells with cells that produce a biological factor for treatingdiseases caused by a deficiency thereof, e.g., encapsulating islet cellswhich produce insulin with Sertoli cells. The use of the co-localized,e.g., encapsulated Sertoli cells and islet cells for treating diabetesmellitus is further described by the present invention. A pharmaceuticalcomposition comprising Sertoli cells and cells that produce a biologicalfactor is also provided.

BACKGROUND OF THE INVENTION

Certain chronic diseases destroy the functional cells in affectedorgans. Mammals with such diseases are often unable to produce proteinsor hormones necessary to maintain homeostasis and usually requirenumerous exogenous substances to survive. Transplanting healthy organsor cells into a mammal suffering from such a disease may be necessary tosave the mammal's life. This type of therapy is generally regarded as alast alternative to curing an otherwise fatal condition. Suchtransplants, however, are often rejected by the body due to an immuneresponse initiated in response to the foreign tissue or cells.Presently, the only recourse to combat this immune response is toadminister chronic nonspecific immunosuppression agents. Unfortunately,this only trades the complications of one chronic disease with othercomplications caused by the immunosuppression agent.

One disease which scientists have attempted to treat with organ and/orcellular transplants but have had very limited success is diabetesmellitus. Diabetes mellitus is a prevalent degenerative disease inmammals. It is characterized by a relative or complete lack of insulinsecretion by the beta cells within the islets of Langerhans of thepancreas or by defective insulin receptors.

This insulin deficiency prevents normal regulation of blood glucoselevels and often leads to hyperglycemia and ketoacidosis. Whenadministered to a mammal, insulin promotes glucose utilization, proteinsynthesis, formation and storage of neutral lipids and the growth ofcertain cell types.

In the United States alone there are approximately 13 million diabetics.Of these, 2.6 million are insulin dependent diabetics. Drug & MarketDev., 4:210 (1994). Health care analysts estimate that diabetes costs$92 billion a year resulting from medical costs and lost productivity.

The various forms of diabetes have been organized into a series ofcategories developed by the National Diabetes Data Group of the NationalInstitutes of Health. Type I diabetes in this classification schemeincludes patients dependent upon insulin to prevent ketosis. This groupof diabetics was previously called juvenile-onset diabetes, brittlediabetes or ketosis-prone diabetes. Type I diabetes is caused by anautoimmune reaction that causes complete destruction of beta cells.

Type II diabetes is classified as adult-onset diabetics. The diabeticpatient may or may not be insulin dependent. Type II diabetes can becaused by a number of factors. For most mammals with Type II diabetes,the beta islet cells are defective in the secretion of insulin.

There are many therapies currently used to treat diabetes, however, eachhas its limitations. The major problem confronting most patients withdiabetes mellitus is that currently available therapies fail to preventthe complications of the disease process. The most common method oftreating Type I diabetes in mammals is providing an endogenous source ofinsulin such as porcine, bovine or human insulin. Insulin injectiontherapy prevents severe hyperglycemia and ketoacidosis, but does notcompletely normalize blood glucose levels. This treatment further failsto prevent the complications of the disease process, including prematurevascular deterioration. Premature vascular deterioration is the leadingcause of morbidity among diabetic patients. Furthermore, complicationsresulting from long-term diabetes include renal failure, retinaldeterioration, angina pectoris, arteriosclerosis, myocardial infarctionand peripheral neuropathy.

A second method of treating diabetes is by transplanting the pancreas inconjunction with the administration of chronic nonspecificimmunosuppression agents. This treatment is usually given to anindividual who has advanced diabetes, such as an individual with kidneyfailure. Whole pancreas transplantation can be successfully done with a75% one year survival rate, but surgical transplantation of the pancreasis very difficult. Furthermore, since the entire organ must be donated,the only practicable source is a deceased donor. In addition, whencyclosporine, the most common immunosuppressive drug used for organtransplants, is administered in a dosage necessary to suppress theimmune response, the drug inhibits pancreatic cell function.Furthermore, the steroids that are often administered with an organtransplant often cause the patient to become diabetic.

A third treatment involves transplanting islet of Langerhans cells intothe diabetic patient. However, islet transplantation has been generallyunsuccessful due to the aggressive immune rejection of islet grafts.(Gray, 1991, Immunology Letters 29:153; Jung et al., 1990, Seminars inSurgical Oncology 6:122). In particular, successful transplantation ofisolated pancreatic islet cells has been very difficult to achieve dueto the chronic administration of immunosuppressive drugs required toprevent organ rejection of the cells following transplantation. Thesedosages of immunosuppressive drugs can cause increased susceptibility toinfection, hypertension, renal failure and tumor growth. Furthermore,unlike most organ transplants, islet cells must grow their own bloodsupply following implantation in the host in order for the cells tosurvive. Conventional transplantation techniques do not provide thenecessary factors to stimulate the production of new blood vessels.

Thus, to successfully transplant cells in a mammal, it is necessary thatthe cellular transplants are not rejected by the recipient and have thecapacity to grow upon transplantation. As a commercial reality, it isfurther necessary that a sufficient quantity of cells are available fortransplantation. Traditionally, the number of cellular transplants havebeen limited by the inability to adequately collect and store asufficient number of cells for transplantation. Conventional storagetechniques, such as cryopreservation, often damage a large quantity ofthe stored cells. Porcine islet cells, for example, are extremelyfragile and easily dissociate into fragments or single cells uponthawing.

The present invention alleviates many of the problems associated withthe current therapies for chronic diseases that destroy the functionalcells of vital organs. Specifically, the present invention provides amethod of creating systemic tolerance to subsequent transplants in themammal. Furthermore, the present invention solves the problemsassociated with the conventional therapies for diabetes mellitus, byproviding a method of transplanting pancreatic islets cells into adiabetic mammal, whereby the cellular transplants produce insulin in thediabetic mammal. The present inventor has previously demonstratedextended functional survival of islet cells allografts and xenografts inthe testis. (Selawry et al., 1989, Diabetes 38:220.) It has beensurprisingly discovered in accordance with the present invention that animmunologically privileged site can be created in a mammal bytransplanting Sertoli cells to a nontesticular site in a mammal. Thenewly created immunologically privileged site allows the transplantationand survival of cells that produce biological factors useful in thetreatment of diseases, especially diabetes. In addition to creating animmunologically privileged site, the Sertoli cells produce cellstimulatory factors which enhance the maturation, proliferation andfunctional capacity of cells. Sertoli cells have further been found toenhance the recovery rate and viability of mammalian cells stored bytechniques such as cryopreservation.

SUMMARY OF THE INVENTION

The present invention relates to a method of treating a disease thatresults from a deficiency of a biological factor in a mammal whichcomprises administering Sertoli cells and cells that produce thebiological factor. In a preferred embodiment, the biological factor is ahormone.

In a more preferred embodiment, the disease is diabetes mellitus, thefactor producing cells are pancreatic islet cells and the factor isinsulin.

In yet another embodiment the cells that produce the biological factorsare cells that have been genetically engineered, for example bytransformation with a nucleic acid that expresses the biological factor.

The present invention further relates to a method of treating diabetesmellitus in a mammal comprising administering pancreatic islet cells andSertoli cells. In a preferred embodiment the Sertoli cells and isletcells are administered by transplantation. The Sertoli cells may beisolated from a mammal or they may be derived from a Sertoli cell line,in accordance with the present invention.

Another aspect of this invention is directed to a method of creating animmunologically privileged site and producing cell stimulatory factorsin a mammal.

A further aspect of the present invention is directed to a method ofcreating systemic tolerance to a subsequent transplant in a mammal bytransplanting Sertoli cells prior to said subsequent transplant.

Still a further aspect of the present invention provides a method ofenhancing the maturation, proliferation and functional capacity of cellsin tissue culture by co-culturing these cells with Sertoli cells.

A method of enhancing the recovery rate and viability of frozenmammalian cells and in particular factor producing cells, in tissueculture by co-culturing these cells with Sertoli cells is furtherprovided by the invention described herein.

Another aspect of the present invention is directed to a method ofco-localizing, e.g., encapsulating the biological factor producingcells, e.g., islet cells, with Sertoli cells and to the use of theco-localized product for enhancing long-term immunoprotection andnutritional survival of islets and for the treatment of diabetes.

Yet another embodiment of the present invention provides apharmaceutical composition comprising Sertoli cells and cells thatproduce a biological factor. In a preferred embodiment thepharmaceutical composition comprises Sertoli cells and pancreatic isletcells and a pharmaceutically acceptable carrier.

The present invention further provides a compartmentalized kitcontaining Sertoli cells and cells that produce a biological factor. Anarticle of manufacture comprising a packaging material and Sertoli cellscontained within the packaging is also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the glucose responses to oral sustacal tolerance tests doneon the monkey "Lucky" at intervals before pancreatectomy (Lucky-pre);after pancreatectomy but prior to transplantations (Lucky-post); and atintervals following transplantation (143 days, 730 days and 930 days,respectively).

FIG. 2 shows the C-peptide responses to an oral sustacal tolerance testat the same time intervals as depicted in FIG. 1.

FIG. 3 shows the glucose responses to oral sustacal tolerance tests inthe monkey "Oscar".

FIG. 4 shows the C-peptide responses in the same animal and at the sameintervals depicted for FIG. 3.

FIGS. 5a and 5b show the effect of intratesticular islet allografts onserum glucose levels and the insulin responses to oral glucose inspontaneously diabetic BB/Wor dp rats. FIG. 5a shows the plasma glucose(mg/dl) concentrations in response to the oral glucose administration of2 g/kg of a 50% glucose solution in three groups of rats: untreatedcontrol Sprague Dawley, transplanted diabetic BB/Wor dp, and insulintreated diabetic BB/Wor dp rats. FIG. 5b shows the serum insulin levelsin response to the same dose of oral glucose in untreated controlSprague Dawley, and in transplanted BB/Wor dp rats.

FIGS. 6a and 6b show the effect of intratesticular islet allografts onplasma glucagon secretory responses to oral glucose and a combination ofglucose plus glipizide in spontaneously diabetic BB/Wor dp rats. FIG. 6ashows the plasma glucagon responses to the oral administration of 2 g/kgof a 50% glucose solution in three groups of rats: untreated controlSprague Dawley, transplanted diabetic BB/Wor dp, and insulin treateddiabetic BB/Wor dp rats. FIG. 6b shows the plasma glucagon responses tothe oral administration of 7 mg/kg of glipizide and 2 g/kg of a 50%glucose solution, administered 30 minutes later, in three groups ofrats: untreated control Sprague Dawley, transplanted diabetic BB/Wor dp,and insulin treated diabetic BB/Wor dp rats. Data points are mean±SE ofeight animals in each group.

FIG. 7 shows a light micrograph of the pancreatic islets of Langerhansand the isolated rat Sertoli cells transplanted into the renalsubcapsular space of a diabetic rat.

FIG. 8 shows an electron micrograph of an individual cell within thetransplanted islet.

FIG. 9 shows an electron micrograph of the fine structure of theextra-islet cells labeled "S" in FIG. 7.

FIG. 10 shows the effect of transplantation of piglet islets and Sertolicells underneath the renal capsule on the mean daily urine output ofseven grafted female rat recipients. Each bar represents the mean dailyurine output over a ten-day period following transplantation.

FIG. 11 shows the effect of the transplantation of piglet islets andSertoli cells underneath the skin on the mean daily urine volumes ofthree rats over a 50-day period.

FIG. 12 shows the light photomicrograph of pig islets of Langerhans andrat Sertoli cells transplanted into the renal subcapsular space of adiabetic rat. IL shows the presence of islands of beta cells (IL)surrounded by an infiltration of small lymphocytes underneath the renalcapsule (K); B (upper left) shows at higher magnification that theislands (IL) consist of beta cells and B (lower right) shows that betacells contain characteristic insulin granules.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a method of treating a disease thatresults from a deficiency of a biological factor in mammals whichcomprises administering to a mammal Sertoli cells and a therapeuticallyeffective amount of cells that produce the biological factor. As definedby the present invention, a biological factor is a protein or nonproteincompound that is necessary for cellular metabolism and homeostasis. In apreferred embodiment, the biological factor is a hormone. Hormoneproducing cells which can be administered using the method described inthe present invention include, for example, pancreatic islet ofLangerhans, pituitary, liver, parathyroid, thyroid and ovarian cells.

In accordance with the present invention, the Sertoli cells and thecells that produce the biological factor can be from the same species asthe mammal to be treated or from a different species. Further, theSertoli cells and the cells that produce the biological factor need notbe derived from the same species. It has been demonstrated in accordancewith the present invention that Sertoli cells from pigs in conjunctionwith islet of Langerhans from pigs can be used in the treatment ofdiabetes mellitus in rats. In a preferred embodiment the Sertoli cellsare bovine, porcine or human.

Sertoli cells, which are the predominant cells of male testes, used inthe method described by the present invention can be separated fromother testicular cells such as Leydig cells, peritubular cells and germcells, using conventional techniques. For example, the testes of a malemammal, such as a boar or ram, are first collected by castration. Thetestes are then chopped into several pieces and subsequently washed bycentrifugation.

Testicular Leydig cells can be removed from the tissue suspension usingdigestion agents such as trypsin and DNase. The remaining cellsuspension is then washed by centrifugation several times. The pellet isresuspended in collagenase, incubated and washed by centrifugation toeliminate peritubular cells within the testes. Testicular germ cells canbe removed by incubating the pellet with hyaluronidase and DNase. Afterseveral washings by centrifugation, the Sertoli cells are collected totransplant using the method of the present invention.

In accordance with the present invention, the Sertoli cells may beobtained by various methodologies which establish a line of cellsderived from primary cultures of mammalian Sertoli cells. In oneembodiment the Sertoli cells are immortalized with a chemical or viraltransformant, e.g., a temperature-sensitive mutant of the SV40 virusthat allows propagation and promotes differentiation of the cells. Inanother embodiment Sertoli cells are isolated from mammalian tissue byconventional means using various hydrolytic enzymes such as collagenase,hyaluronidase, and the like. The cells are further isolated from thetissue by such conventional methods as filtering and centrifugation toobtain a purified Sertoli cell population. The isolated and purifiedSertoli cells are next incubated and conventionally immortalized underconditions known in the art such as treating said cells with a chemical,that transforms the DNA thereof, e.g. a mutagen. Examples includeN-nitrosylmethylureas, nitrous acid, hypoxanthine, nitrosamines (see,Freshney, I. R. in Culture of Animal Cells, A Manual of Basic Technique,3 ed., Chapter 15, Wiley-Liss, New York). Alternately, the isolatedpurified sertoli cells are incubated in a virus-containing mediumconsisting of, e.g., SV40 virus or polyoma virus and a conventionalgrowth medium such as F12/DMEM, for sufficient time to propagate theSertoli cells, which are then isolated from the virus. If an infectiousvirus cell is being utilized, then it is preferred that the virus beattenuated by techniques known in the art. The Sertoli cells may beisolated from the virus or chemical by conventional techniques employinghydrolytic enzymes. To verify that the Sertoli cells are produced bythis methodology, the isolated Sertoli cells are optionally screened forthe expression of an appropriate isolate for cloning, e.g., on the basisof expression of mRNAs encoding Sertoli cell-secreted proteins.

In accordance with the present invention, a biological factor is aprotein or nonprotein compound that is absent, deficient or altered in adisease state. Cells that produce a biological factor can be isolated,for example, by first surgically removing the tissue that produces thefactor from a mammal. This tissue is subsequently chopped and digestedusing conventional techniques. For example, the tissue can be digestedusing a collagenase digestion. The particular factor producing cells cansubsequently be collected from the digestion mixture using a separationgradient such as a Ficoll gradient. The factor producing cells are thengrown in tissue culture in serum using conventional techniques.

In accordance with the present invention, the factor producing cells maybe co-cultured with Sertoli cells in tissue culture. Furthermore,factor-producing mammalian cells may be co-cultured, co-localized orco-transplanted with Sertoli cells to enhance the maturation,proliferation and functional capacity of the mammalian cells. It hasbeen demonstrated in accordance with the present invention that thematuration of porcine islet cells was enhanced when these cells wereco-cultured with Sertoli cells as evidenced by both the structuralintegrity and functionality of the porcine islet cells compared to theislet cells cultured without Sertoli cells. Thus, maturation is definedby the present invention as the process by which a cell develops andbecomes functional. The enhanced proliferation of porcine islet cellsco-cultured with Sertoli cells is evidenced by the larger number ofviable, insulin producing cells compared to porcine islet cells culturedwithout Sertoli cells. Proliferation as used herein, is defined as aprocess in which cells multiply. The enhanced functional capacity ofporcine islet cells cultured with Sertoli cells is evidenced by thegreater capacity of the co-cultured islet cells to respond to glucoseand glucose plus Forskolin as insulin secretagogues. Functional capacityis defined as the ability of a cell to respond the biologicalenvironment and to generate various chemical and biological substancesin response to the various substances present in the biologicalenvironment (e.g. when islet cells produce insulin in the presence ofglucose).

Mammalian cells which can be co-cultured, co-localized orco-transplanted with Sertoli cells as described by the present inventioninclude, for example, germ cells, such as sperm cells, oocytes, ovariancells and zygotes; endocrine cells, such as pancreatic islet cells,chromaffin, thyroid cells, hepatocytes, parathyroid cells, Leydig cells,follicular cells; hybridoma cells; recombinantly transformed cells;epithelial cells; nerve cells and epidermal cells. In a preferredembodiment, the mammalian cell is a germ cell or endocrine cell. Cellsgrown in tissue culture can be transplanted into a mammal in conjunctionwith the Sertoli cells using the methods of the present invention. Inaccordance with the present invention, factor producing cells may bestored using a variety of conventional techniques, such ascryopreserving the cells prior to growth in tissue culture forsubsequent transplantation. It has been observed in accordance with thepresent invention, that Sertoli cells co-cultured, co-localized orco-transplanted with mammalian cells, and in particular factor producingcells such as islet cells, enhance the recovery rate and viability ofthe mammalian cells in tissue culture and in particular, enhance therecovery rate and viability of cells that have been previously storedusing techniques such as cryopreservation. Moreover, when co-localizedwith factor producing cells, such as islet cells, Sertoli cells provideimmunoprotection and nutritional support when the two cell types areproximally located. Sertoli cells protect the factor producing cells,such as islets, from, inter alia, macrophages, proteins, lymphokines(e.g., IL-1) and toxic factors released by activated lymphocytes.Sertoli cells provide nutritional support for islets through Sertolisecreted growth factors, e.g., IGF (insulin-like growth factor), EGF(epidermal growth factor) and transferrin, thereby permitting thefactor-producing cells to survive longer than when the Sertoli cells arenot present.

In a preferred embodiment the factor is a hormone, and the hormoneproducing cells are isolated from a tissue source as described above.For example, insulin-producing cells are isolated from the pancreas. Inanother preferred embodiment, the factor producing cells are provided bytransforming suitable host cells with a nucleic acid capable ofexpressing the factor of interest. Transformed cells are provided bymethods known to one of ordinary skill in the art, and can be found in amyriad of textbooks and laboratory mammals, including Sambrook et al.(1989) Molecular Cloning: A Laboratory Mammal, Cold Spring HarborLaboratories, Cold Spring, N.Y. If necessary, the nucleic acid encodingthe factor of interest can be adapted by methods known to one ofordinary skill in the art to effect secretion of the factor from thetransformed cell. The utilization of Sertoli cells in conjunction withthe factor producing cells in accordance with the method of the presentinvention allows the production of an immunologically privileged siteand production of cell stimulatory factors in the treated mammal.

The administration of factor producing cells and Sertoli cells into amammal is accomplished by conventional techniques. In a preferredembodiment, administration is by transplantation and the factorproducing cells are injected into the mammal concurrently with orimmediately after the injection of the Sertoli cells into the same site.In another embodiment, Sertoli cells and cells producing the biologicalfactor are co-localized and administered to a mammal. As an example,islets and Sertoli cells are co-encapsulated and injected into themammal intraperitoneally. In another embodiment, co-localized islets andSertoli cells are transplanted, injected or provided into a biologicalor non-biological biocompatible material (biomaterial). Examples of abiological biocompatible material include an isolated segment of smallintestine with intact circulation, a pouch (e.g. an omental pouch or agastric pouch etc.), a biocompatible polymeric scaffold, a polymericsponge or matrix, and the like, prepared pursuant to conventionaltechniques. On the other hand, a non-biological, biocompatible materialincludes reticulated thermoplastics such as acylnitrile vinyl chloridecopolymer (PAN-PVC), and the like. The biomaterial may be conventionallyimplanted in a mammal. In accordance with the present invention, anexogenous biological factor may be administered following thetransplantation of factor producing cells and Sertoli cells until thetransplanted cells produce a therapeutically effective amount of thebiological factor. For the treatment of diabetes, for example, insulinmay be administered following the transplantation of pancreatic isletcells and Sertoli cells until the transplanted islet cells produce atherapeutically effective amount of insulin.

The Sertoli cells and factor producing cells of the present inventioncan be transplanted or co-localized using any technique capable ofintroducing the cells into the mammal such as parenteral administration,subcutaneous administration following surgical exposure to a desiredsite, biocompatible scaffold, sponge or matrix delivery orintraperitoneal administration. Prior to transplantation, the recipientmammal is anesthetized using local or general anesthesia according toconventional technique. In a preferred embodiment the mammal to betreated is human. In another embodiment the present method of treatingdisease further comprises administering an immunosuppressive agent suchas, for example, cyclosporine, tacrolimus, despergualin and monoclonalantibodies to, e.g., T cells. In a preferred embodiment theimmunosuppressive agent is cyclosporine. In another preferred embodimentcyclosporine is administered at a dosage of from 0.5 mg to 200 mg/kgbody weight. In a most preferred embodiment cyclosporine is administeredat a dosage of from 5 mg to 40 mg/kg body weight.

It has been discovered in accordance with the present invention thatadministration of Sertoli cells results in the creation of animmunologically privileged site in the treated mammal and in theproduction of cell stimulatory factors. An immunologically privilegedsite as defined by the present invention is a site in the mammal wherethe immune response produced in response to the transplanted cells issuppressed due to immunosuppressive agents produced by Sertoli cells.Immunologically privileged sites are characterized by an available bloodsupply to provide nourishment for the transplanted cells and a densetissue to keep the transplanted cells within close proximity of eachother. Examples of immunologically privileged sites as defined by thepresent invention include the renal subcapsular space, subcutaneousfacie, the brain and the hepatic portal vein. Cell stimulatory factorsare defined by the present invention as factors that enhance theviability of mammalian cells. For example, it has been shown inaccordance with the present invention that Sertoli cells increase therate at which the transplanted factor producing cells vascularize in thetransplanted site (i.e. promote angiogenesis). Further, it has beenshown by the present invention that these cell stimulatory factorsenhance the maturation, proliferation and functional capacity of cellstransplanted with Sertoli cells. It is therefore indicated that theSertoli cells produce cell stimulatory factors which enhance ofviability of mammalian cells as evidence, for example, by the increasedvascularization rate of the transplanted islet cells. As used herein,viability denotes the number of living cells in a preparation.

In a preferred embodiment, the present invention describes a method oftreating diabetes mellitus by transplanting islet of Langerhans inconjunction with Sertoli cells to create an immunologically privilegedsite. Allografts as used in the present invention describes the transferof tissues or cells between two genetically dissimilar mammals of thesame species. The term xenografts in the present invention describes thetransfer of tissues or cells between two mammals of different species.

The transplanted islet of Langerhans cells and Sertoli cells used in themethod described by the present invention can be prepared using anynumber of conventional techniques. For example, islet of Langerhanscells can be prepared from the pancreas of several mammals of the samespecies. The pancreases are pooled together, chopped up and digestedusing collagenase. The islet of Langerhans cells can be further isolatedusing conventional gradients. Once isolated, the islet cells can begrown in culture and then transplanted in conjunction with Sertoli cellsto create an immunoprivileged site.

Sertoli cells used in the method described by the present invention canbe derived from primary cultures of mammalian Sertoli cells according tothe methods known to one skilled in the art including the method of e.g.Roberts et al. (1995) Biology of Reprod. 53:1446-1453, the contents ofwhich is incorporated herein by reference, or the Sertoli cells can beisolated from mammalian male testes. To collect the islet cells, thetestes are first chopped into several pieces and then washed bycentrifugation. Leydig cells, present in the crude mixture, can beremoved from the tissue suspension using digestion agents such astrypsin and DNase. The remaining cell suspension is then washed bycentrifugation several times. Following, the pellet may be resuspendedin collagenase, incubated and washed by centrifugation to eliminateperitubular cells within the testes. Testicular germ cells can beremoved by incubating the pellet with hyaluronidase and DNase. Afterseveral washings by centrifugation, the Sertoli cells fortransplantation can be collected.

The Sertoli cells can be transplanted to create an immunoprivileged sitewithin a mammal using a variety of techniques. For example, after themammal is anesthetized, the Sertoli cells can be injected into a tissuemass, thereby creating an immunoprivileged site. The Sertoli cells andfactor producing cells of the present invention can be combined usingthe techniques capable of co-localizing the cells such asmicroencapsulation inside biocompatible membranes, hydrogels, orreticulated thermoplastics, for example. The co-localized cells cansubsequently be injected, transplanted or provided into a tissue masssubcutaneously or into a pouch, e.g. an intestinal pouch, an omentalpouch, a gastric pouch, or a biocompatible polymeric scaffold, sponge ormatrix consisting of, e.g., polylacetic acid. Once injected,transplanted, or provided, the co-localized product is used to treatdiseases caused by a deficiency of the biological factor. For example,the Sertoli cells and islet cells in combination are useful in treatingdiabetes mellitus.

Sertoli cells are administered in an amount effective to provide animmunologically privileged site. Such an effective amount is defined asthat which prevents immune rejection of the subsequently orco-administered cells that produce the biological factor. Immunerejection can be determined for example histologically, or by functionalassessment of the factor produced by the cells.

The present invention further provides a method of creating systemictolerance to a subsequent transplant in a mammal by transplantingSertoli cells prior to said subsequent transplant as described herein. Atransplant as used herein is a mammalian cell, tissue, organelle ororgan that is removed from one mammal and placed in the same ordifferent mammal. The subsequent transplant may be made in the same siteor a secondary site. A secondary site as used herein, is atransplantation site in the mammal different from the initialtransplantation site. Systemic tolerance is demonstrated by variousbiological phenomena. For example, systemic tolerance results in adiminished destructive immune response to a subsequent allograft orxenograft in a mammal without the administration of prolongedimmunosuppressive agents or the co-transplantation of Sertoli cells. Inaccordance with the present invention, the allograft or xenograft may beany type of transplant, including cells, tissues, organelles or anorgan. The types of cells which may be transplanted in accordance withthe methods described by the present invention include, for example,endocrine cells, bone marrow cells, hepatocytes or liver cells, nervecells or brain cells, and islet cells (fetal, neonatal or adult). Thetypes of tissues, organelles or organs which may be transplanted inaccordance with the methods described by the present invention include,for example, heart, kidney, pancreas, liver, skin, ligaments, tendonsand cartilage.

As demonstrated by the present invention, a mammal may be tolerized(i.e. systemic tolerance may be achieved) by a variety of procedures.For example, systemic tolerance may be achieved by transplanting anallograft or xenograft with Sertoli cells and subsequently transplantingthe same type of allograft or xenograft without Sertoli cells or aprolonged administration of immunosuppressive agents. Systemic tolerancemay also be achieved by transplanting an allograft of any cell, tissue,organelle or organ without Sertoli cells or prolonged immunosuppressiveagents following an initial transplantation of an allograft with Sertolicells. In a preferred embodiment Sertoli cells are administered inamounts ranging from 10¹ to 10¹⁰ cells. In a more preferred embodiment,10⁵ to 10¹⁰ cells are administered.

The cells producing the biological factor are administered in atherapeutically effective amount. The ordinary skilled artisan candetermine the appropriate amount of cells producing the biologicalfactor by methods known in the art. The amount of cells is dependentupon the amount of factor being produced by the cells and the knowntherapeutically effective amount of the factor necessary to treat thedisease. For example, 1 to 1000 islet cells per gram body weight can beadministered to treat diabetes using allografts, 20 to 1000 islets pergram body weight are administered using xenografts. In another preferredembodiment, 5 to 100 islet cells per gram body weight are administeredto treat diabetes. In a most preferred embodiment, 5 to 20 islet cellsper gram body weight are administered, using allografts and 100-1000islet cells per gram body weight are administered for xenografts.

In another embodiment the present method of treating diabetes furthercomprises administering an immunosuppressive agent such as, for example,cyclosporine, tacrolimus, despergualin and monoclonal antibodies to,e.g., T cells. In a preferred embodiment the immunosuppressive agent iscyclosporine. In another preferred embodiment cyclosporine isadministered at a dosage of from 0.5 mg to 200 mg/kg body weight. In amost preferred embodiment cyclosporine is administered at a dosage offrom 5 mg to 40 mg/kg body weight.

More generally, the immunosuppressive agent can be administered for atime sufficient to permit the transplanted islets to be functional. Thisperiod extends from the point prior to or immediately following thetransplantation of the islets to the point at which the cells arecapable of producing therapeutically effective amounts of insulin. In apreferred embodiment, the sufficient period of time to administer animmunosuppressive agent is about 40 to about 100 days followingtransplantation of the islets. In a more preferred embodiment, thesufficient period of time is about 50-60 days.

A preferred embodiment of this invention is directed to a method oftreating Type I and Type II diabetes mellitus by transplanting islet ofLangerhans in conjunction with Sertoli cells into the renal subcapsularspace.

Unlike the therapies for diabetes described in the prior art, the methodof treating diabetes described by the present invention prevents thecomplications of the disease process and does not result in the adverseside effects associated with conventional diabetes therapy. Furthermore,the method of transplanting islet cells described by the presentinvention provides the necessary factors for angiogenesis, growthenhancing and increased functional capacity of the islet transplants.

A method of creating an immunologically privileged site in a mammal isfurther described by the present invention. An immunologicallyprivileged site is created by transplanting isolated Sertoli cells intoa mammal in an amount effective to create an immunologically privilegedsite. In a preferred embodiment, 10¹ to 10¹⁰ cells are administered. Ina more preferred embodiment, 10⁵ to 10¹⁰ cells are administered. In apreferred embodiment the Sertoli cells are transplanted into the renalsubcapsular space or subcutaneous facie by injection. In a preferredembodiment the mammal is a human and the Sertoli cells are human orporcine.

A further aspect of the present invention is directed to a method ofenhancing the recovery rate and viability of frozen mammalian cells intissue culture comprising co-culturing the frozen mammalian cell withSertoli cells. As shown in accordance with the present invention,Sertoli cells produce cell stimulatory factors which enhance therecovery rate and viability of mammalian cells previously frozen.Mammalian cells may be frozen using a widely conventional techniques,including, for example, cryopreservation.

Further contemplated in accordance with the present invention is amethod of enhancing the recovery and proliferation of ex vivo cellscomprising co-culturing said cells with Sertoli cells for a time andunder conditions sufficient to achieve said enhanced recovery andproliferation.

Another aspect of the present invention provides a pharmaceuticalcomposition comprising Sertoli cells and cells producing a biologicalfactor and a pharmaceutically acceptable carrier. In a preferredembodiment the composition comprises Sertoli cells and islet ofLangerhans cells and a pharmaceutically acceptable carrier. A furtherpreferred embodiment of the present invention comprises using porcine,bovine or human Sertoli cells and porcine, bovine or human islet ofLangerhans cells. As used herein, a pharmaceutically acceptable carrierincludes any and all biological and non-biological biocompatiblemembrane materials. A pharmaceutically acceptable carrier also includesany conventional solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic agents and the like. The use of such mediaand agents is well-known in the art.

The present invention further contemplates a pharmaceutical compositioncomprising Sertoli cells and a pharmaceutically acceptable carrier. Thispharmaceutical composition, upon administration to a mammal, can be usedto treat a variety of diseases, such as for example, autoimmunediseases. Accordingly, the present invention is further directed totreating an autoimmune disease in a mammal comprising administering atherapeutically effective amount of Sertoli cells to the mammal.

The present invention is further directed to a method of enhancing therecovery and proliferation of ex vivo cells comprising culturing saidcells with a culture media from a tissue culture containing Sertolicells for a time and under conditions sufficient to achieve saidenhanced recovery and proliferation. As contemplated by the presentinvention, Sertoli cells are cultured using a conventional tissueculture media as described herein for a time and under conditionssufficient for the Sertoli cells to produce, for example, cellstimulatory factors. The Sertoli cells are then removed from the culturemedia and the culture media is used in subsequent tissue cultures, forexample, as a culture media for sperm cells previously stored bycryopreservation.

Another aspect of the present invention is directed to methods ofco-localizing biological factor producing cells, e.g., islets ofLangerhans, with Sertoli cells to enhance long-term immunoprotection andnutritional survival of transplanted factor producing cells, e.g.,islets. The methods of co-localization include co-localization in anintestinal segment, pouch, e.g. an omental pouch, a gastric pouch orbiocompatible polymeric scaffold, sponge or matrix, for example. Themethod of co-localization in an intestinal segment comprises:

(a) isolating a segment of mammalian small intestine with intact mucosaand intact circulation;

(b) removing the mucosal layer of the small intestine and closing theends of the isolated segment;

(c) implanting biological factor producing cells and Sertoli cells intothe isolated segment; and

(d) fixing the isolated segment to the small intestine.

Methods of co-localizing biological factor producing cells, e.g., isletswith Sertoli cells in pouches, such as omental or gastric pouches arealso contemplated by the present invention as described by Amiri, et al.(1990) Arch. Surg. 125:1472-1474 and Bayat, et al. (1995) Surg. Res.Commun. 17:87-91, both of which are incorporated herein by reference.Procedures for co-localizing islets and Sertoli cells in biologicallycompatible pouches are general and conventionally employed on acase-by-case basis by the skilled artisan in accordance with the presentinvention.

Procedures for co-localizing islets and Sertoli cells in polymericscaffolds are readily appreciated by the skilled artisan. It ispreferred that the polymeric templates used in accordance with thepresent invention are biodegradable and comprise a polyvinyl alcohol,e.g., poly-L-lactic acid. Polyvinyl alcohol based templates providesufficient porosity to permit rapid tissue ingrowth andprevascularization before cell transplantation. In essence, thepolymeric template employed in connection with the present inventionacts as a matrix or sponge permitting the ingrowth of blood vessels andtissue which facilitates the co-localization of, e.g., islets andSertoli cells by providing ready access to a nutrient-rich blood supply.

It is preferred that the Sertoli cells are provided in an amount rangingfrom about 10¹ to about 10¹⁰ cells. In a more preferred embodiment,Sertoli cells are provided in an amount ranging from about 10⁴ to about10¹⁰ cells. In yet another preferred embodiment, the factor producingcells are pancreatic islets. The islets are provided in a preferredamount of about 5 to about 200 cells per gram of body weight, and in amore preferred amount of about 5 to about 100 cells per gram of bodyweight.

A further aspect of the present invention is directed to a method ofencapsulating biological factor producing cells, e.g., islets, withSertoli cells to enhance long-term immunoprotection and nutritionalsurvival of transplanted islets. The method of encapsulation comprises:

(a) suspending a pharmaceutically effective amount of biological factorproducing cells, e.g., islets, and Sertoli cells in combination with agelling effective amount of a first water soluble gelling agent in anaqueous medium which is physiologically compatible with the cells andextruding the islet/Sertoli cell/gelling agent mixture to form a dropletcontaining the islets and Sertoli cells;

(b) subjecting the product of step (a) to an effective amount of networkforming cations to form discrete capsules of sufficient size toencapsulate the islets and Sertoli cells together;

(c) forming a semipermeable membrane around the capsules to obtain asingle-walled bead encapsulating the cells; and

(d) contacting the single-walled bead with a gelling effective amount ofa second gelling agent so as to form a second semi-permeable membraneencapsulating the product of step (c).

The gelling agent may be any water-soluble material which can be gelledto form a bead. A preferred gelling agent is a water soluble, natural orsynthetic polysaccharide gum such as an alkali metal alginate. Apreferred gum is sodium alginate. Other gums which may be used includeguar gum, gum arabic, carrageenan, pectin, tragacanth gum, xanthan gumor their acidic fractions.

In a preferred embodiment, the Sertoli cells and factor producing cellsare encapsulated within an alginate polylysine-alginate semi-permeablehydrogel. It is preferred that the Sertoli cells are derived frombovine, porcine, and human sources and are produced by a cell line inaccordance with the present invention. It is preferred that the Sertolicells are provided in an amount of from 10¹ to 10¹⁰ cells. In a morepreferred embodiment, Sertoli cells are provided in an amount of fromabout 10⁴ to 10¹⁰ cells. In yet another preferred embodiment the factorproducing cells are pancreatic islets. The islets are provided in apreferred amount of about 5 to about 200 cells per gram of body weight,and in a more preferred amount of about 5 to about 100 cells per gram ofbody weight.

The procedure for the encapsulating of Sertoli cells with cells thatproduce a biological factor is general, and the procedure will beexplained in more detail with respect to Sertoli cells and islet cells,which are exemplary.

In an embodiment of step (a) of the subject method, the first gellingagent is sodium alginate. It is conventionally suspended in an aqueoussolution such as a buffer or saline solution containing the islets andSertoli cells.

By "gelling effective amount" is meant an amount of a water solublegelling agent capable of binding calcium ions or other ions thatinteract with the gelling agent to form a network. More specifically,the islets and Sertoli cells in combination are preferably suspendedrelative to the gelling agent in a ratio of about 1:20 to 20:1 (v/v) andmore preferably 1:10 to 10:1 (v/v) and most preferably about 1:10 (v/v).Preferably, the Sertoli cells and islets cells are present in a ratio ofabout 1:1 (v/v).

The suspension is extruded by techniques commonly used in the art. It ispreferred that the suspension is extruded through an air-jet needle. Ina preferred embodiment, droplets containing islets and Sertoli cells inassociation with the alginate are produced by extrusion (1.7 ml/min)through a 22 gauge air-jet needle (air flow 5 l/min).

In an embodiment of step (b) of the subject method, the droplets aresubjected to a solution of multivalent cations, such as a solution ofcalcium salt, e.g., calcium halide, e.g., calcium chloride solution,which form a network within said droplet. The preferred concentration isat least about 0.5% (v/v), and more preferably at least about 1% (v/v),and most preferably ranging from about 1% (v/v) to a saturated solution.In an embodiment, the droplets fall into a beaker containing a salinesolution at pH7 of calcium chloride solution, e.g., 10 ml 1.1% CaCl₂ in0.9% saline at pH7. This process continues for a sufficient time untilthe negatively charged alginate droplets bind calcium and form calciumalginate gel.

In an embodiment of step (c) of the subject method, a membrane is formedaround the product of step (b) by subjecting the encapsulated product topolymers, which polymers contain substituents reactive with the gellingagents, especially the acid groups of the gelling agent. The preferredpolymers are polyamine acids such as poly-L-lysine (PLL) orpolyethylenimine. In a preferred embodiment, the polymer ispoly-L-lysine with a molecular weight of about 20 kd. It is preferredthat the polymers coat the product of (b) by following the procedure ofGoosen, et al. in Biotech. Bioeng., 27: 146-150 (1985), the contents ofwhich is incorporated herein by reference. Without wishing to be bound,it is believed that positively charged poly-L-lysine displaces calciumions and binds negatively charged alginate, producing a polyelectrolytemembrane.

In an embodiment of step (d) of the subject method the second gellingagent may be sodium alginate which may improve the biocompatability ofthe capsule in a mammal. The second gelling agent may be added accordingto the methods employed by Weber, et al. U.S. Pat. No. 5,227,298,incorporated herein by reference. The double walled semi-permeablecapsules formulated in connection with the present invention havemolecular weight cut-offs in the range of 50,000 daltons and providesustained release of factors produced by the encapsulated islets andSertoli cells. The capsules comprise semi-permeable membranes whichfunction to protect islets from immune responses while simultaneouslypermitting passage of biological factors produced by islets into themammal. In still another embodiment of the present invention,co-localized, e.g., co-encapsulated islets and Sertoli cells areconnected to a blood supply by techniques known in the art, therebypermitting the free flow of nutrients and inhibiting the influx ofmolecules produced by the immune system.

The co-localized, e.g., encapsulated cells producing biological factorand Sertoli cells are effective in treating a disease resulting from adeficiency of said biological factor. For example, the co-localized,e.g., encapsulated islet cells with Sertoli cells, are effective intreating diabetes mellitus. Thus a preferred embodiment of thisinvention is directed to a method of treating diabetes mellitus byco-localizing, e.g., co-encapsulating and transplanting islet ofLangerhans into the peritoneal space. This method not only prevents thecomplications of the disease process, but also reduces the adverseeffects associated with other therapies. This method also provides abiological factor in appropriate amounts which are released in aphysiological manner.

The present invention is also directed to a kit for treatment of adisease. In one embodiment, the kit is compartmentalized to receive afirst container adapted to contain Sertoli cells in an amount effectiveto create an immunologically privileged site in a mammal, and a secondcontainer adapted to contain a therapeutically effective amount of cellsthat produce a biological factor that is absent or defective in thedisease to be treated. In a preferred embodiment, the Sertoli cells arebovine, porcine or human and are provided in an amount of from 10¹ to10¹⁰ cells. In a more preferred embodiment, Sertoli cells are providedin an amount of from 10⁵ to 10¹⁰ cells. In another preferred embodimentthe cells that produce a biological factor are cells that have beentransformed with DNA encoding the factor. In yet another preferredembodiment the cells that produce the factor are pancreatic islet cells.The islet cells are provided in a preferred amount of 5 to 200 cells pergram of body weight, and in a more preferred amount of 5 to 100 cellsper gram of body weight.

The present invention further provides an article of manufacturecomprising a packaging material and Sertoli cells contained within saidpackaging material, wherein said Sertoli cells are effective forcreating an immunologically privileged site in a mammal, and whereinsaid packaging material contains a label that indicates that saidSertoli cells can be used for creating an immunologically privilegedsite in a mammal. The packaging material used to contain the Sertolicells can comprise glass, plastic, metal or any other suitably inertmaterial.

Unless specified to the contrary, it is to be understood thatpercentages are by volume.

In order to further illustrate the present invention, the experimentsdescribed in the following examples were carried out. It should beunderstood that the invention is not limited to the specific examples orthe details described therein. The results obtained from the experimentsdescribed in the examples are shown in the accompanying figures andtables.

EXAMPLE 1

Six male Rhesus monkeys were transplanted with islet allografts in theirtestes to examine the survival of these transplants. The recipients weremade diabetic by means of a near total pancreatectomy, followed twoweeks later by an intravenous injection of 35 mg streptozotocin/kg bodyweight. This procedure resulted in the induction of severe diabetesmelitis. Plasma glucose levels were in excess of 400 mg/dl and theanimals were ketotic. Malabsorption was prevented by the oraladministration of VACUOUS®, one tablet given twice daily before eachmeal.

Islets were isolated from female Rhesus monkeys. First, the pancreasesof five animals were removed, pooled and chopped finely into smallerfragments. After collagenase digestion in a water bath at 37° C., theislets were separated from exocrine tissues and other cellular debris onat least two Ficoll gradients, prepared in tandem. The islets werewashed three times by centrifugation in ice-cold Hanks's buffer and thenhandpicked and transferred in groups of 150 to biologic grade Petridishes. Each dish contained 6 mL of culture medium CMRL-1066supplemented with 5% fetal calf serum, glucose at a concentration of 250mg/dL, penicillin (100 U/mL), and streptomycin (100 μ/mL). Incubation ofislets were carried out at 35° C. in 5% CO and air for 4 to 6 days. Theislets were transferred to fresh medium at 48 hour intervals.

Viability and counting of the islets were facilitated by means of theuptake of the dye dithizone. Each monkey received an average of about10⁴ islets/kg body weight injected into both testes. In the first threeanimals the testes were elevated into the abdominal cavity, whereas inthe last three recipients the grafted organs were anchored into theinguinal canal. Cyclosporine (CsA) was administered, in varying doses tothe first three grafted animals over a 30 day period, whereas the lastthree hosts were given 7 injections of CsA (20 mg/kg) on days -4 to +3.Oral sustacal tolerance tests were done on day 30, and then at intervalsin the normoglycemic animals, as follows.

The monkeys were housed individually in cages and given standard monkeychow and fruit twice daily. In addition, a pancreatic enzyme was mixedwith the food since the monkeys had been pancreatectomized to make themdiabetic before transplantation.

The night before the test, the animals were fasted for 12 hours. At 8a.m. the next morning they were then anesthetized and prepared for thetest meal. Sustacal was used as the test agent. Sustacal consists of aphysiologic mixture of carbohydrates, proteins and fat which closelymimics a standard meal and which is a powerful stimulus for the releaseof insulin.

Sustacal was injected directly into the stomach of the sleeping animalthrough a nasogastric tube. Blood samples were then obtained at times 0,15, 30, 60, 90, 120 and 180 minutes. The samples were centrifuged andthe serum stored at -20° C. until measurements for insulin or C-peptidecould be carried out. C-peptide is a very sensitive marker for beta cellfunction. The results are shown in FIGS. 1-4.

FIG. 1 shows the glucose responses to oral sustacal tolerance tests doneon the monkey "Lucky" at intervals before pancreatectomy (Lucky-pre);after pancreatectomy but prior to transplantation (Lucky-post); and atintervals following transplantation (143 days, 730 days and 930 days,respectively).

It can be readily appreciated that the animal became severely diabeticafter the removal of his pancreas (Lucky-post). Followingtransplantation the glucose responses were restored to normal levels atall of the time intervals measured (143, 730 and 930 days followingtransplantation). Lucky showed no evidence of graft failure. With graftfailure glucose levels would become elevated would approach those whichwere found following his pancreatectomy.

FIG. 2 shows the C-peptide responses to an oral sustacal tolerance testat the same time intervals as depicted in FIG. 1. Following hispancreatectomy the C-peptide responses became blunted indicating asevere diabetes. But following transplantation the levels were not onlyrestored to normal but appeared to show a hyperresponsive pattern ofC-peptide release and levels done on day 730 exceed the normal levels atall points measured. The elevated levels might be due to the fact thatinsulin released from the testis enters the systemic circulation. Bycontrast, insulin released from the pancreas enters the portal vein andtravels immediately to the liver where about 60% is broken down duringthe first passage. Insulin released into the systemic circulationreaches the liver much later, thus the elevated levels. As was evidentwith an investigation of the glucose concentrations, the C-peptideresponses showed no evidence of failure 30 months followingtransplantation.

FIG. 3 shows the glucose responses to oral sustacal tolerance tests inthe monkey "Oscar". Following the removal of his pancreas he becameseverely diabetic with elevated glucose levels. Followingtransplantation of islets the glucose responses became similar to thosedetermined before his pancreas was removed. The glucose levels remainwithin normal levels 32 months following transplantation.

FIG. 4 shows the C-peptide responses in the same animal and at the sameintervals depicted for FIG. 3. The animal became very diabetic followingthe removal of his pancreas and shows blunted C-peptide responses as aresult. Following transplantation and for the next 730 days theC-peptide responses were greater compared with the normals. on day 930following transplantation the C-peptide responses have become somewhatless compared with the normals. Despite somewhat lower C-peptide levelsthe animal remains normoglycemic.

This example demonstrates that primates can be successfully transplantedwith intratesticular islet allografts without the need for sustainedimmunosuppression, and that functional integrity of intratesticularislet allografts is maintained for periods exceeding two years with noevidence of graft failure.

EXAMPLE 2

This study examined insulin and glucagon secretory patterns inspontaneously diabetic bb/Wor dp rats transplanted with abdominal,intratesticular, islet grafts. Diabetic, BB/Wor dp, rats receivedintratesticular islet grafts from MHC-compatible BB/Wor dr rats and noimmunosuppression. After a period of 74±15 days, of normoglycdmia, threedifferent groups (controls; BB/Wor dp, transplanted; and BB/Wor dp,insulin treated) were given the following challenges; (1) an oralglucose tolerance test (OGTT), (2) a single oral dose of glipizide,followed by an OGTT, and (3) arginine, by intravenous infusion. Theresults of this study are shown in Tables 1 and 2 and FIGS. 5 and 6.

                  TABLE 1    ______________________________________    Metabolic Parameters and Immunoreactive Serum    Insulin and Glucagon Levels in Control and in    Transplanted and Insulin Treated BB/Wor dp Rats                BB/Wor dp                                  INSULIN                CONTROLS                        GRAFTED*  TREATED    ______________________________________    Plasma Glucose (mg/dl):                  112 ± 5                            502 ± 8+                                      510 ± 13+    Prior to Therapy    After 2.5 Months                  97 ± 4 110 ± 3                                      350 ± 40#    Duration p.t. OGTT (days)                  75 ± 6 70 ± 11                                      78 ± 19    Weight Gain (g)                  120 ± 6                            105 ± 17                                      48 ± 14$    Fasting Plasma Insulin                  21.9 ± 3                            20.4 ± 2                                      ND    (uU/ml)    Fasting Plasma Glucagon                  37.8 ± 5.7                            43.4 ± 4.6                                      47.4 ± 4.9    (pg/ml)    ______________________________________     *Duration of normoglycemia after grafting (days) = 279 ± 25     + P < 0.0001 vs. control     # P < 0.0001 vs. grafted     $ P < 0.02 vs. grafted

                  TABLE 2    ______________________________________    Pancreatic and Testicular Insulin    and Glucagon Content in Control and in    Transplanted and Insulin Treated BB/Wor dp Rats              BB/Wor dp                                 INSULIN              CONTROLS GRAFTED   TREATED    ______________________________________    Pancreas (mg)                1573 ± 171                           757 ± 122                                     920 ± 32    Insulin (ug/g)                66 ± 5.03                           0.58 ± 0.18                                     0.76 ± 0.12    Glucagon (ng/mg)                4.1 ± 0.35*                           49. ± 0.33**                                     6.9 ± 0.08    Testes Fractions: (mg)                493 ± 49.6                           582 ± 59.2                                     430 ± 28.0    Insulin (ug/g)                0.0        59.70 ± 0.49                                     0.0    Glucagon (ng/mg)                0.0        1.4 ± 0.37                                     0.0    ______________________________________     *P < 0.03     **P < 0.08 vs. diabetic, respectively

FIG. 5 shows the effect of intratesticular islet allografts on serumglucose and insulin responses to oral glucose in spontaneously diabeticBB/Wor dp rats. FIG. 6 shows the effect of intratesticular isletallografts on plasma glucagon secretory responses to oral glucose and acombination of glucose plus glipizide in spontaneously diabetic BB/Wordp rats. This experiment demonstrates that grafted testes inspontaneously diabetic BB/Wor dp rats contain both alpha and beta cells,and that the alpha and beta cells have the capacity to respond tospecific secretagogues independently.

EXAMPLE 3

This study investigated the effect of Sertoli cell enriched fraction(SEF) on islet allograft survival in the renal subcapsular space ofdiabetic rats.

The animals used in this study were PVG rats, weighing between 150-200g. Diabetes was induced by means of a single intravenous injection of 65mg/dL of streptozotocin. Only rats with plasma glucose levels in excessof 400 mg/dL were transplanted. Sprague Dawley (S-D) outbred rats wereused as islet donors. Either PVG or S-D male rats between 16 and 18 daysold were used as Sertoli cell donors.

Islet Preparation

Islets were prepared according to modification of the method of Londonet al. (1990) Transplantation, 49: 1109-1113. The islets were purifiedon Ficoll gradients, and the isolated cells were then incubated for 4days at 37° C. in a humidified atmosphere of 5% CO₂, and air prior touse. No special efforts were made to deplete the islets of contaminatingpassenger leukocytes.

Sertoli Cell-enriched Fraction Preparation

Highly purified preparations of Sertoli cells were isolated form thetestes of young males according to the method of Cheng et al. J. Biol.Chem., 26:12768-12779. The testes were removed, chopped into severalpieces, and placed in a 50 mL conical tube containing 50 mL of Ham'sF12/DMEM media. The pieces were washed once by centrifugation at 800×gfor 2 min. The supernatant was aspirated, and the tissue resuspended in40 mL of media containing 40 mg trypsin and 0.8 mg DNase in a sterile250 mL Erlenmeyer flask. The flask was placed in 37° C. oscillatingincubator at 60-90 osc/min for 30 min. This step removed Leydig cells.The tubules were then transferred to a 50 mL conical tube, andcentrifuged at 800×g for 2 min. The supernatant fraction was aspirated,and the pellet resuspended in 40 mL of 1 M glycine, 2 mM EDTA containing0.01% soy bean trypsin inhibitor and 0.8 mg DNase, and incubated at roomtemperature for 10 min. This step lysed any residual Leydig cells. Thecells were washed by centrifugation for 2 min, and the step repeatedtwice, or until the media was no longer cloudy. The pellet wasresuspended by gentle homogenization with a glass Pasteur pipet in 40 mLof media containing 20 mg collagenase in an Erlenmeyer flask, andincubated at 37° C. for 5 min with 60-90 osc/min. The cell suspensionwas centrifuged at 800×g for two min, and the pellet resuspended bygentle homogenization with a Pasteur pipet in 40 mL media containing 40mg collagenase and 0.2 mg DNase, and incubated in an Erlenmeyer flask at37° C. for 30 min with 60-90 osc/min. The cells were then washed bycentrifugation for 2 min, and the process repeated at least three timesto eliminate peritubular cells. The, cells were resuspended by gentlehomogenization with a Pasteur pipet in 40 mL media containing 40 mghyaluronidase and 0.2 mg of DNase, and incubated at 37° C. for 30 minwith 60-90 osc/min. The cells were pelleted by soft centrifugation for 2min, and washed at least five times to eliminate germ cells. Theresultant SEF was resuspended in 0.25 mL of media, and immediatelytransplanted into the recipient rat. Each grafted rat received theequivalent of the total amount of Sertoli cells contained in a singletestis.

Transplantation of Rats

The diabetic rat was anesthetized with methoxyflurane USP in a sterilehood and the left flank opened to expose the kidney. TheSertoli-enriched fraction containing approximately 5 million Sertolicells was injected first underneath the renal capsule. The cells couldbe seen as a milkish bubble underneath the capsule. Immediatelyafterwards, a total of 10 islets/g of body weight was injected to thesame milkish bubble. The needle was retracted slowly to prevent leakageof the grafted cells. Cyclosporine (CsA) was administered subcutaneouslyin varying doses over a 20-day period to groups two and four. Becausethe grafted rats responded similarly whether the drug was administeredover a 20-day, or over a 3-day period, all of the subsequent groups,including the female rats, were treated with only three injections of 25mg/kg CsA, given on days 0, +1, and +2, relative to the graft. The ratsreceived no other therapy.

A total of 36 male and 21 female PVG rats were divided into sixdifferent treatment groups: Group 1, the control group, consisted of 6male rats grafted with only islets from S-D donor rats. They receivedneither SEF nor CsA. Group 2 consisted of 10 rats grafted with acombination of islets from S-D rats and CsA postransplantation, but noSEF. Group 3 consisted of a total of 10 rats grafted with a combinationof islets from S-D and SEF from PVG donor rats, but no CsApostransplantation. Group 4 consisted of 10 rats grafted with acombination of islets from S-D donors, SEF from PVG donors, and CsApostransplantation. Group 5 consisted of 11 female rats grafted with thesame combination of cells as depicted for Group four. Group 6 consistedof 10 female rats grafted with a combination of islets and SEF, bothcell types from S-D donors, and CsA postransplantation.

Posttransplantation Evaluation of Rats

The grafted rats were transferred to metabolic cages, and plasma glucoselevels were obtained at weekly intervals. Urine volumes and urineglucose contents were obtained at daily intervals. A rat was consideredcured of the diabetic process if the following criteria were met: Arandom plasma glucose level ≦150 mg/DL; glycosuria; and immediatereversal to hyperglycemia following surgical removal of the graftedkidney.

To determine if any of the rats had become unresponsive to their grafts,normoglycemic rats were challenged with a secondary islet allograftconsisting of at least 500, freshly prepared, Sprague Dawley isletswhich were injected into the contralateral renal subcapsular space. Noimmunosuppression was given following the challenge.

To examine the impact of the transplantation of SEF on fertility of thefemale rats, normoglycemic animals of longer than 30 days were matedwith PVG males. Metabolic parameters, as outlined above, were closelymonitored, as was the course of their pregnancies.

Structural Analysis of Grafted Tissue

A total of five successfully grafted rats were nephrectomized atintervals following transplantation. Wedge sections of renal tissue,obtained from sites at which islets and SEF had been injected, wereprepared for examination by light and electron microscopy, as previouslydescribed by Cameron et al. (1990) Transplantation, 50:649-653. Briefly,the tissue wedges were immersion-fixed with 5% glutaraldehyde in 0.1 Mcollidine buffer for 1 h, washed in buffer, and postfixed for 1 h with1% osmium tetroxide in 0.1 M buffer. Small tissue blocks were cut fromthe wedges, and dehydrated through a graded series of ethyl alcohols,transferred to propylene oxide, and embedded in Epon 812/Aralditeplastic resin. Thick (0.5 μm) and thin (900 mg) sections were strainedroutinely with toluidine blue and uranil acetate/lead citrate,respectively, for structural analysis by light and electron microscopy.The results are shown in Table 3 and FIGS. 7-9.

                  TABLE 3    ______________________________________    Effect of Sertoli Cells on    Islet Allograft Survival in the    Non-Immunologically Privileged Renal, Subcapsular Site                                  Duration of    Group         Sertoli Cell    Normoglycemia (days)    (n)   Gender  (donor origin)                             CsA  Individual Responses    ______________________________________    1 (6) Male    -          --   0, 0, 0, 0, 0, 0    2 (10)          Male    --         +    0, 0, 0, 0, 0, 0, 0, 130 > 441,                                  >445    3 (10)          Male    +(PVG)     -    0, 0, 0, 0, 9, 10, 12, 13,                                  13, 14    4 (10)          Male    +(PVG)     +    19, 76, 58*, 84*, 167*, 127†,                                  139†, >418†, >422 ,                                  >425†    5 (11)          Female  +(PVG)     +    7, 11, 14, 28, >287†, >305†,                                  >306†, >308†, >441†,                                  >447†,                                  >457†    6 (10)          Female  +(S-D)     +    8, 10, 96*, 128*, >168, >172,                                  >184, >193, >193, >196    ______________________________________     *Nephrectomized     †Challenged with a Secondary Islet Allograft

Group 1: None of the six rats grafted with islets alone, without eitherSEF or CsA, became normoglycemic.

Group 2: Three of 10 rats grafted with islets and treated with CsAbecame normoglycemic for more than 100 days. The 3 normoglycemic ratswere challenged with a secondary graft on days 116, 192 and 197,respectively. One rat reverted to hyperglycemia on day 130, while 2remained normoglycemic.

Group 3: Initially 6 of the 10 rats grafted with islets and SEF, but noCsA, became normoglycemic, but all of them reverted to hyperglycemia byday 14.

Group 4: All 10 of rats grafted with a combination of SEF and islets,and also given CsA became normoglycemic. Two reverted spontaneously todiabetes on days 19 and 76, respectively. Three were nephrectomized ondays 58, 84 and 167 following transplantation. All 3 of these ratsbecame hyperglycemic within the next 24 h. The remaining 5 rats werechallenged with a secondary islet allograft on days 119, 129, 280 342and 400, respectively. Of these, the first 2 reverted to diabetes on day127 and 139, respectively, while the latter 3 remained normoglycemic.

Group 5: All 11 of the female rats grafted with a combination of isletsand SEF, and then given CsA, became normoglycemic. of these, 4 revertedspontaneously to hyperglycemia by day 28. Of the 7 normoglycemic ratswho were mated with male PVG rats, 6 became pregnant, and of these, 8had litters varying between 1 and 10 pups. They were able to nurse thepups successfully. A total of 7 of the long-term surviving females werechallenged with secondary islet allografts at least 200 days followingtransplantation. None of them reverted to hyperglycemia.

Group 6: of the 10 rats grafted with islets and SEF from the same donorstrain of rat, all 10 became normoglycemic. Two reverted tohyperglycemia by day 10. A nephrectomy to remove the graft was done on 2of the long-term surviving rats on days 96 and 201, respectively. Bothreverted to hyperglycemic immediately within the next 24 h.

Tissue Morphology

Renal tissue obtained from the long-term grafted kidney appearedstructurally normal by light microscopy (FIG. 7). Transplanted islets inthis organ were immediately subjacent to the kidney capsule, and alsoappeared structurally normal. They displayed tissue and cellulararchitecture identical to islets in situ (FIG. 7). Individual isletcells were partitioned into cell clusters by thin connective septacontaining small vessels and capillaries (FIG. 7). It appeared that mostof the islet cells contained secretion granules. When resolved byelectron microscopy, islet cells were identified as the β-cell type bythe inclusion of ultrastructurally distinctive, and uniqueinsulin-containing secretion granules (FIG. 8). All β-cell clustersobserved were in close proximity to intra-islet capillaries (FIG. 8).

There was a high density of cells between, and directly adjacent to, thetransplanted islets and renal parenchyma. By light microscopy, they didnot appear to be islet cells, kidney cells nor cells or blood origin(FIG. 7). When observed by electron microscopy, these cells were similarin ultrastructure to Sertoli cells in that their nucleic were irregularin profile, and contained deep nuclear clefts, distinctive nucleoli wereoften present, and mitochondrial structure was dense. Although thesecells did not retain the typical polarity of Sertoli cells in vivo, theywere, however, identical in appearance to Sertoli cells in vitro, whenthe cells are not plated on a basement membrane substrate. The cellswere not associated with a basement membrane, and appeared randomlyorganized (FIG. 9). Cells showing ultrastructural features of eithergerm or Leydig cells were not observed.

This example demonstrates that an immunologically privileged site fortransplantation of isolated islet can be created in male and femalediabetic recipients by transplantation of Sertoli cells without the needfor sustained immunosuppression.

EXAMPLE 4

This study determined the survival of discordant islet xenografts invarious nonimmunologically privileged organ sites in experimentalanimals.

Islets were prepared from young piglets as follows: Male piglets notweighing more than 2.2 kg were used exclusively. The piglet wasanesthetized and following exsanguination both pancreas and testes wereharvested under sterile conditions. A collagenase solution consisting of2 mg/ml of collagenase type XI (Sigma) was injected directly into thepancreas. The pancreas was incubated at 37° C. for 17 minutes and thedigested tissues washed three times by means of centrifugation andaliquots of 1 ml each transferred to Petri dishes. The islets wereincubated at 32° C. in tissue culture media 199 supplemented with 10%horse serum for six days.

On day seven the cultured islets were collected in batches of ±4,000 andcryopreserved using a standard protocol. The cells were stored in liquidnitrogen at 96° C. for periods varying between two and four weeks. Theislets were removed from the liquid nitrogen and thawed using anestablished procedure. The thawed islets were transferred to Petridishes and co-cultured with pig Sertoli cells for three days at 32° C.in the same 199 culture media as described above. Earlier studies haveshown an improved survival rate of thawed islets cultured in thepresence of Sertoli cells.

On day three following thawing the islets were hand-picked and countedand a total amount of 12 islets/g of body weight transplanted intofemale diabetic Sprague Dawley rats. A total of 5 million Sertoli cellsprocured from the piglet testes were grafted simultaneously into thesame location. The organ sites to be tested for the grafting of isletsinclude: a! the renal subcapsular space, b! subcutaneously, and c! theliver. Following transplantation, the rats were treated withcyclosporine as follows: 25 mg/kg for 7 days: 15 mg/kg for 5 days; 10mg/kg for 5 days; 5 mg/kg for an additional 13 days. On day 30 the drugwas discontinued.

To demonstrate viability and functional integrity of isolated pigletislets the following studies were done: a) staining of Cells withdithizone, a stain is highly specific for insulin; b) staining of cellswith 0.4% trypan blue which indicates viability of the islets; and c)culturing of batches of 5 islets in the presence of insulinsecretagogues such as low and high glucose concentrations at specifiedintervals following culturing, cryopreservation and thawing. The resultsare shown in Table 4.

                  TABLE 4    ______________________________________    Insulin Secretion (micro-units/ml) from    Incubated and from Cryopreserved-Thawed Islets    Done on Days 3, 7 and 14 of Culturing, Respectively                 3 DAYS  7 DAYS    14 DAYS    ______________________________________    INCUBATED ISLETS PRIOR TO CRYOPRESERVATION:    a) Low Glucose (90 mg/dl)                   15.3 ± 3.8                             21.8 ± 1.1                                       17.29 ± 2.4    b) High Glucose (300 mg/dl)                   32.2 ± 5.4                             37.14 ± 3.4                                       23.3 ± 1.8    CRYOPRESERVED AND THAWED ISLETS:    a) Low Glucose (90 mg/dl)                   14.52 ± 2.8                             7.13 ± 1.3                                       5.38 ± 2.02    b) Low Glucose + Sertoli                   10.31 ± 2.8                             9.17 ± 2.6                                       8.38 ± .41    Cells    ______________________________________

                                      TABLE 5    __________________________________________________________________________    Yield of Porcine Islets    Following 1, 3 and 7 Days of Culture and the    Percentage of Islets Lost During 7 Days of Culture                                       Islet    Pig.        BW   Panc.                  D1 Islets/                         D3 Islets/                                D7 Islets/                                       Loss %    No. (kg) W g  g panc.                         g panc.                                g panc.                                       D7/D1    __________________________________________________________________________    1   1.6  1.79 36,536 31,659 27,212 26%    2   2.0  1.89 37,272 32,962 27,883 25%    3   2.3  2.46 29,268 26,046 20,884 29%    4   1.8  1.66 39,904 37,726 31,664 21%    5   1.8  1.76 37,846 34,578 30,046 21%    6   1.6  1.74 39,866 37,888 32,424 19%    7   1.4  1.61 42,126 39,456 33,872 20%    8   2.3  2.48 33,682 29,334 24,892 26%    9   2.1  2.28 43,478 41,226 37,394 14%    10  2.1  2.09 40,126 36,448 33,282 17%    11  2.1  2.12 31,248 27,170 26,415 15%    12  2.1  1.98 38,848 36,465 29,293 25%    13  2.2  2.06 39,146 37,446 31,709 19%    14  2.2  2.24 27,892 25,028 21,342 23%    15  2.7  2.69 44,610 38,364 31,524 29%    16  1.5  1.44 42,222 40,414 31,244 26%    Mean ±        2.0 ± 0.3             2.0 ± 0.4                  37692 ± 1233                         34513 ± 1307                                29442 ± 1119                                       22.2 ± 1.2%    SE    __________________________________________________________________________

                  TABLE 6    ______________________________________    Recovery of Islets Following Freezing and    Thawing in Presence and Absence of Sertoli Cells    Islets alone            Islets + Sertoli cells    No.     Pre-   Post    Recovery                                  Pre-  Post  Recovery    of islets            cryo   thawing (%)    cryo  thawing                                              (%)    ______________________________________    D3F/D3T 250    152     61%    290   212   73%            230    131     57%    260   228   88%            440    278     63%    430   280   88%            420    366     87%    410   324   79%            450    290     64%    440   358   81%                   Means   66.4%              81.8%    D7F/D3T 260    136     52%    250   229   92%            300    208     69%    300   202   67%            280    177     53%    290   238   82%            360    205     57%    350   300   86%            320    218     68%    390   289   74%            380    217     57%    320   270   84%                   Means   61.0%              80.8%    ______________________________________

As shown in Table 5, the yield of islets per gram pancreas was37692±1233, 34513±1307 and 29,442±1119, after 1, 3 and 7 days ofculture, respectively. Following cryopreservation and thawing andreculturing of the cells in the presence of Sertoli cells approximately20% of the cells were damaged or lost as shown in Table 6. Thus ±24,000islets/gram of piglet pancreas were available for transplant purposesafter cryopreservation and thawing.

The results showed that insulin secretion was blunted when glucose wasused as insulin secretagogue prior to cryopreservation. The effect wasmore evident following cryopreservation and thawing. While the presenceof Sertoli cells had marked effects on number of islets that survivedcryopreservation and thawing their presence had little effect on theability of the islets to respond to a low glucose concentration asinsulin releasing agent. However, as shown in Example 8 the presence ofSertoli cells augmented the secretion of insulin in the presence of highglucose concentrations and glucose plus Forskolin.

EXAMPLE 5 Response of Diabetic Sprague Dawley Rats to theTransplantation of Islets from Piglet Donors (Discordant Xenografts)

The rats were made diabetic by means of a single i.v. injection of 55mg/kg of streptozotocin. They were grafted only if the blood sugar wasequal to or more than 400 mg/dl. Following transplantation the rats wereplaced individually in metabolic cages and urine volume, urine glucosecontent, and body weights were measured at daily intervals. Bloodglucose levels were done at weekly intervals. A rat is considered curedof diabetes if the blood glucose level is 160 mg/dl or less and/or thedaily urine volume is 15 ml or less.

The results are illustrated in FIGS. 10 and 11.

FIG. 10 shows the effect of transplantation of piglet islets and Sertolicells underneath the renal capsule on the mean daily urine output ofseven grafted female rat recipients. Each bar represents the mean dailyurine output over a ten-day period following transplantation. The studyhas been conducted over an 80-day period, the bar on the furthest rightthus showing the mean urine output per day from day 80 through 89, etc.

The figure shows that the mean daily urine volume for the first 60 daysvaried between 19.7 mls and 27 mls or within a diabetic range. It can bereadily appreciated that urine volumes decreased to near-normal levelsonly from days 70 through day 89. The corresponding plasma glucoselevels during the first and last ten day periods were 474±46 and 155±70,mg/dl, respectively.

These results indicate that following transplantation with piglet isletsand Sertoli cells the rats showed evidence of survival of the graftedislets. The reversal to normoglycemia took about 80 days.

It should be noted that one of the cured rats is pregnant and has beennormoglycemic throughout her pregnancy.

FIG. 11 shows the effect of the transplantation of piglet islets andSertoli cells underneath the skin on the mean daily urine volumes ofthree rats over a 50 day period. The results show that the mean urinevolume decreased from a mean of 41.7 ml during the first 10-day periodto an average of 12.3 mls during the fifth week. The correspondingglucose levels were 509±45, and 200±12, mg/dl, respectively.

The data depicted above demonstrate that both the renal subcapsularspace and the subcutaneous area can be used as a site to create animmunologically privileged site for the transplantation of isletxenografts.

EXAMPLE 6

This study determined the effect of cultured Sertoli cells on thesurvival of discordant islet xenografts in diabetic rats with minimalearly exogenous immunosuppression.

Preparation of Islets

Neonatal piglets of less than seven days of age were killed byanesthesia and islets were isolated according to a method of Kuo C. Y.,Burghen G. A., Myvacle A. and Herrod H. G. (1994) "Isolation of isletsfrom neonatal pig pancreatic tissue", J. Tissue Culture Methods, 16:1-7. Briefly, the pancreas was distended by an injection of acollagenase solution, 2 mg/ml, collagenase type Xl, in culture mediumDMEM. After incubation at 39° C. for 17 min, the digested fragments werewashed by centrifugation and the digested tissue was then incubated forone week in medium 199 supplemented with 10% horse serum and 1%antibiotics at 32° C. The islets were then cryopreserved according tothe method by Lakey J. R. T., Warnock G. L., Kneteman N. M., Ao Z.,Rajotte R. V. (1994) "Effects of precryopreservation culture on humanislet recovery and in vitro function", Transplant Proc., 26:820 andstored in liquid nitrogen at -196° C. Three days prior totransplantation the cryopreserved islets were rapidly thawed andcultured at 32° C. for two days. One day prior to transplantation someof the islets were collected and co-cultured with Sertoli cells for 24hours.

Sertoli cell isolation

Testes of young S-D rats were removed and Sertoli cells were isolated bythe method of Cheng C. Y. and Bardin C. W. (1987) "Identification of twotestosteroneresponsive proteins in Sertoli cell-enriched culture mediumwhose secretion is suppressed by cells of the intact seminiferoustubule." J. Biol. Chem., 262:12768-12779. Briefly, the testes weredigested first in DMEM containing 1.0% trypsin, and then is DMEMcontaining 1.0% collagenase, type 1, for periods of 15 min each, at 37°C. The purified Sertoli cells were cultured at 37° C. in DMEM/F12supplemented with transferrin, 10 ug/ml, FSH 10 ng/ml, insulin 20 ug/mland 1.0% FCS, for three days. For transplantation, Sertoli cells andislets were pooled and rats were grafted with either a compositeconsisting of 5×10⁶ Sertoli cells and 3,000 islets, or with islets alone(15 islets/g of body weight).

Transplantation of rats

Female S-D rats, weighing between 170 and 200 g were made diabetic bymeans of a single i.v. injection of 60 mg/kg of streptozotocin. A totalof 31 diabetic rats were divided into 3 groups and grafted as follows:Group 1, a control group (n=8), received a total of 15 islets/g bodyweight injected underneath the renal capsule. No Sertoli cells weregrafted. Following transplantation the rats were treated withcyclosporine for 55 days: 25 mg/kg for 3 days, 15 mg/kg for 10 days, 10mg/kg for 10 days and 5 mg/kg for the following 32 days.Immunosuppression was then stopped. Each rat received, in addition, 1-3U of Ultralente insulin at daily intervals if the 24-hour urine glucosecontent exceeded I g. Insulin therapy was stopped on day 55. Group 1, atissue control group (n=8), was given a renal, subcapsular injection ofa composite of about 5×106 Sertoli cells and 3,000 islets. No CsA wasgiven. Insulin was given as depicted above. Group 3, the experimentalgroup (n=15), was transplanted with both sertoli cells and islets andthen treated with CsA and insulin according to the schedule outlinedabove.

Posttransplantation evaluation of rats

Plasma glucose levels were obtained at weekly intervals. Twenty fourhour urine volumes and urine glucose contents were recorded daily. A ratwas considered cured of the diabetic process if the following criteriaapplied: A plasma glucose level of equal to or less than 10 mmol/L, a24-hour urine volume of less than 15 ml, and immediate reversal tohyperglycemia following surgical removal of the grafted kidney. Onenormoglycemic rat was mated on day 69 to test her ability to becomepregnant.

Structural analysis of the grafted tissue

Two normoglycemic rats were nephrectomized on days 117 and 330 andgrafted tissue prepared for light and electron microscopy. Selawry H.P., Cameron D. F. (1992) "Sertoli cell-enriched fractions in successfulislet cell-transplantation", Cell Trans., 2:123-129. Briefly, tissuewedges were immersion-fixed with 5% glutaraldehyde in 0.1 M collidinebuffer for 1 h., washed in buffer, and postfixed for 1 h with 1% osmiumtetroxide in 0.1 M buffer. Small tissue blocks were cut from the wedges,and dehydrated through a graded series of ethyl alcohols, transferred topropylene oxide, and embedded in Epon 812/Araldite plastic resin. Thick(0.5 um) and thin (900 ng) sections were stained routinely withtoluidine blue and urinal acetate/lead citrate, respectively, forstructural analysis by light and electron microscopy.

The results of the effect of Sertoli cells and cyclosporine on survivalof xenographic transplantation of pig islet cells into the renalsubcapsular space of diabetic female rats are shown in Table 7.

                  TABLE 7    ______________________________________    Group    Sertoli          Graft Survival    (n)      Cells   CsA      (days)    ______________________________________    1 (8)    -       =        0, 0, 0, 0, 0, 0, 0, 0    2 (8)    +       -        0, 0, 0, 0, 0, 0, 0, 0    3 (15)   +       +        0, 0, 0, 0, 0, 71, 77, 96, 117*                              148#, >154, >165, >327, 330*    ______________________________________     *rats nephrectomized to remove the xenograft     #rat died during a cardiac puncture

As shown in Table 7, none of the rats grafted with islets alone and thengiven CsA and low-dose insulin (Group 1) became significantly lesshyperglycemic. Further, none of the rats grafted with a composite ofislets and Sertoli cells, but without CsA, showed any improvement ofhyperglycemia (Group 2). Of 15 rats grafted with islets and Sertolicells and then given CsA (Group 3), 10 showed evidence of reversal ofthe diabetic state. Four of the ten are still normoglycemic for periodsof more than 154, 165, 165, and 327 days, respectively. Thenormoglycemic rats who were nephrectomized on days 117 and 330, becamehyperglycemic immediately. Their plasma glucose levels were 4.9 mmol/L,and 8.2 mmol/L, prior to, and 20.7 mmol/L, and 32.2 mmol/L,respectively, following nephrectomy. A female rat who was mated on day69 became pregnant and delivered a total of 10 pups on day 89, all ofwhom she nursed successfully while remaining normoglycemic. She died onday 148 as a result of a cardiac puncture. Three of 10 rats regressedinto hyperglycemia on days 71, 77, and 96, respectively, after a shortperiod of euglycemia.

These results demonstrate that prolonged survival of a discordant isletxenograft (pig to rat) can be achieved in female diabetic rats. Survivalof islet xenografts depended upon two factors which had to beadministered concomitantly: Co-transplantation with Sertoli cells andtreatment with cyclosporine.

The response of total urine volumes following transplantation with acomposite of pig islet and rat Sertoli cells measured at 10-dayintervals over an 80 day period for 7 of the improved rats showed anaverage daily urine volume of 27.0±13.0 ml/rat during the first 10-dayperiod, which slowly declined to a mean of 12.0±4.0 ml/rat, 70 daysfollowing transplantation.

Tissue morphology studies shown in FIG. 12 show that the tissue andcellular structure of kidney parenchyma appeared normal in the ratnephrectomized 117 days following transplantation. Normal appearingislets with structurally distinct B-cells were visible in wellvascularized areas subjacent to the kidney capsule. Additionally, normalappearing Sertoli cells were observed adjacent to the transplantedislets along with numerous lymphocytes. No plasma cells were identifiedat the transplantation site. Viable endocrine cells were similarlyobserved in the subcapsular renal space of the rat nephrectomized 330days following transplantation.

These studies show that significant prolongation of survival of adiscordant islet xenograft can be achieved without sustainedimmunosuppression. These studies demonstrate that the mechanism by whichSertoli cells promote islet xenograft survival is three-fold: (1)Sertoli cells stimulate the recovery of islets damaged duringtransplantation (i.e. improve the yield and function of culturedislets), (2) Sertoli cells protect grafted islets from immunologicrejection by producing factors which strongly suppress proliferation ofT-cells, and (3) Sertoli cells protect grafted islets from the toxiceffects of cyclosporine.

EXAMPLE 7

This study shows a method of isolating and cryopreserving porcinepancreatic islets for future xenographic transplants in mammals.

Male piglets, <7 days old and weighing 2± kg were used as donors. Thepancreases, weighing 1.4±0.3 g, were harvested and injected with DMEMsolution containing 2 mg/ml collagenase XI. The distended pancreas wasincubated in a shaking water bath at 39° C. for 17 min. The digestedtissue was filtered through a 500 μm stainless steal filter andfiltrates were washed×3 with cold DMEM. Without further purification thecells were cultured in M199 and 10% horse serum at 32° C. for 7 days.The islet cells were then cryopreserved using standard procedures. Atspecified intervals islets were thawed and cultured in M199, both inpresence, and isolated from testes of male piglets according to astandard method.

To test functional capacity, islets cultured for 3 and 7 days wereassessed for insulin release in static incubation. In separateexperiments, effect of insulin secretagogues was tested on isletscultured with and without Sertoli cells. The results of this study areshown in Tables 8 and 9.

                  TABLE 8    ______________________________________    Effect of Insulin Secretagogues,    Glucose and Glucose Plus Forskolin,    on Insulin Release From Incubated and    Frozen/Thawed (F/T) Islets in the Presence    and Absence of Pig Sertoli Cells                 Insulin Release (uU/ml/10 islets)           3.3 mmol/L                   16.7 mmol/L                              16.7 mmol/L glucose           glucose glucose    +100 umol Forskolin    ______________________________________    Day 3    42.3 ± 1.2                       112.8 ± 17.7*#                                  267.7 ± 43.0**#    Incubated with    Sertoli cells    Day 3    31.3 ± 2.1                       57.3 ± 3.8*                                  123.4 ± 15.3**    Incubated    alone    Day 7    22.9 ± 1.9                       64.5 ± 6.4*#                                  153.9 ± 14.6**    Incubated with    Sertoli cells    Day 7    21.3 ± 1.2                       37.3 ± 6.0*                                  120.3 ± 11.4**    Incubated    alone    Day 3 F/T with             20.6 ± 4.3                       44.9 ± 9.9*                                  77.1 ± 13.7**    Sertoli cells    Day 3 F/T             11.7 ± 2.3                       27.9 ± 6.6*                                  54.5 ± 10.7**    alone    ______________________________________     Anova Test: *vs 3.3 mmol/L p 0.5, **vs both 3.3 & 16.7 mmol/L P < 0.05     #with Sertoli cells vs islets alone P < 0.05

                  TABLE 9    ______________________________________    Effect of Sertoli cells on insulin content of incubated and    frozen-thawed piglet islets.    Insulin content (uU/10 islet(s)                Islets alone                          Islets & Sertoli cells    ______________________________________    Incubated D1  257.0 ± 19.6                              391.1 ± 51.4*    Incubated D3  201.1 ± 19.1#                              400.1 ± 41.0*#    Incubated D7  179.1 ± 26.2#                              271.9 ± 39.9*#    Frozen D3/Thaw D3                  52.4 ± 10.3                              132.5 ± 35.1    Frozen D7/Thaw D3                  10.4 ± 0.9                              35.1 + 8.2    ______________________________________     Anova *islets +Sertoli cell vs. islet alone P < 0.05     #Incubated islets D3, D7 vs. Frozen D3, D7 P < 0.05

These results show that: (1) large numbers of neonatal porcine isletscan be isolated by a simple method; (2) cryopreservation and thawingresults in about 40% loss in number of islets in the absence of Sertolicells and about a 20% loss in the presence of Sertoli cells; (3)cultured islets have the ability to respond to both glucose andglucose+Forskolin as insulin secretagogues; (4) the functional capacityof the cocultured islet was enhanced two-fold in the presence of Sertolicells; (5) following cryopreservation and thawing, islets recover morerapidly in presence of Sertoli cells and the response to both glucoseand glucose+Forskolin was enhanced two fold in the presence of Sertolicells.

EXAMPLE 8

This example describes a method of treating genetic diabetes which isdemonstrated using the animal model NOD (non-Obese Diabetic).

Genetic diabetic mice (NOD) are recipients of pancreatic isletxenografts. Diabetic mice are selected from the colony of NOD micemaintained at the University of Tennessee Medical Center. The currentincidence of diabetes in this colony is 80% for females and 63% of malesby 25 weeks of age. Animals are considered diabetic if they have twoconsecutive weekly urine glucose readings of 1/2% (3+) and aconfirmatory plasma glucose greater than 400 mg/dl. Three to ten daysbefore transplantation of the graft, the animals receive appropriateinsulin to stabilize their health. Diabetic animals are randomly dividedinto two groups. All animals in these two groups receive 0.3 mg of theantiCD4 antibody, GK1.5 on days -1, 0 and 1 to initiateimmunosuppression. Maintenance immunosuppression with GK1.5 cyclosporineA, FK506, cyclophosphamide, rapamycin, nicotinamide or15-deoxyspergualin may be required.

Porcine pancreatic islets for transplantation are prepared as describedin Example 6 except that they are not cryopreserved. Nicotinamide (10mM) or IGF-1 may be added to the incubation medium prior totransplantation. Porcine sertoli cells for transplant are prepared asdescribed in Example 6. Cyclosporine may be included in the culturemedium during the first four incubation days prior to transplantation.

One group of the diabetic NOD mice receive 3,000 porcine islets in 25 μlof Hank's buffered salt solution (HBSS) under the right renal capsulefollowed by an injection of 2×10⁷ pig Sertoli cells in 25 μl in HBSSunder the same renal capsule. A second group of mice receive atransplant consisting of only the porcine islet cells. Animals withxenografts continue to receive daily insulin injections; the amountdetermined by the concentration of glucose in the animals urine andplasma. Mice with plasma glucose levels less than 250 mg/dl areconsidered cured and no additional insulin administered.

A majority of mice receiving porcine sertoli cells and porcinepancreatic islets attain normal urine and plasma glucose levels(xenograft acceptors). In contrast, the majority of mice receivingporcine islets alone exhibit graft failure and do not attain normalglucose levels in the urine or plasma.

EXAMPLE 9

This example shows a method of monitoring the immune response elicitedagainst cellular transplants.

Total spleen leukocytes are isolated from mice by methods known in theart from mice that have rejected their porcine islet xenografts. Inpreliminary experiments these leukocytes are administered to NOD/scidmice (NOD mice that in addition have genetic immunodeficiency) withsuccessful pig islet xenografts. The leukocytes cause rejection of thexenograft.

Plastic adherence (to deplete macrophages), nylon wool adherence (todeplete non-T lymphocytes), or specific antibodies (anti-CD4, anti-CD8,anti-F4/80, anti-B220) are used to deplete the total splenocytepreparations of certain classes of leukocytes. The class-depletedleukocyte preparations are injected into the NOD/scid mice withsuccessful pig islet xenografts to determine which leukocyte class isnecessary and sufficient to cause xenograft rejection.

Spleen leukocytes are isolated by conventional methods known in the artfrom mice (xenograft acceptor of Example 8) that have accepted their pigislet xenografts. These leukocytes are administered to NOC/scid micewith successful pig islet xenografts. The leukocytes do not causerejection of the xenograft. Combining the appropriate leukocyte-depletedpreparation that causes rejection with leukocytes from a xenograftacceptor of Example 8 (50/50 mixture) and administering the mixed cellpopulation to NOD/scid xenograft recipients (adoptive transfer) allowsdetermination of whether so-called suppressor lymphocytes are preventingxenograft rejection in xenograft acceptors.

EXAMPLE 10

This example provides a method of encapsulation of Sertoli cells withislets for transplantation.

Preparation of Islets and Sertoli Cell Isolation

Islets are isolated from female Fischer rats and are incubated in CMRLmedium for approximately four days prior to usage. Sertoli cells areisolated from weanling male Fischer rats and are incubated forapproximately four days to confluency in Petri dishes at 37° C.

After four days of incubation, the islets are counted and groups of 50islets are transferred to 24 well Petri dishes. Sertoli cells areremoved from Petri dishes with Sigma non-enzymatic media. The Sertolicells are washed and counted.

Three experimental groups are then established as follows: Group 1:12-well Petri dishes, each well containing 50 islets; Group 2: 12-wellPetri dishes, each well containing a total of 1×10⁴ Sertoli cells; andGroup 3: 12-well Petri dishes, each well containing 50 islets, plus1×10⁴ Sertoli cells. The Petri dishes are incubated at 37° C. for 24hours to permit Sertoli cells to attach to islets.

Microencapsulation of Islets and Sertoli Cells

Islets, Sertoli cells and islets plus Sertoli cells are encapsulated bysuspension in a solution of sodium alginate which is sprayed into a dishof calcium chloride using a droplet forming device according to themethod of Lim et al. (1980 Science 210:908-910, the contents of which isincorporated by reference. The droplets are coated with a layer ofpoly-L-lysine (PLL) with an average size of 20 kDa at a concentration of0.05% (w/v) and a reaction time of 6 minutes according to the method ofGoosen et al. (1985) Biotechnol. Bioeng. 27:146-150, the contents ofwhich is incorporated by reference. An additional outer layer of sodiumalginate is added around the capsule according to the method of O'Sheaet al. (1984) Biochim. Biophys. Acta 804:133-136, incorporated herein byreference. Alternatively, isolated cells may be encapsulated accordingto the methodology of Weber et al. U.S. Pat. No. 5,227,298, incorporatedherein by reference. Following encapsulation, the cells are divided intotreatment groups. Group 1: Free islets, not encapsulated, Group 2:Islets alone, encapsulated, Group 3: Sertoli cells alone, encapsulated,and Group 4: Islets plus Sertoli cells encapsulated. Encapsulated cellsare placed in media conventionally selected by the skilled artisan at37° C.

In Vitro Encapsulation

At specified intervals following encapsulation, i.e., 1, 7, 14, 21 and30 days respectively, following incubation, functionality of the groupscontaining islets are examined. Approximately 10 capsules from each ofthe islet containing groups are stimulated, in tandem, by a bufferedmedium containing glucose 9 mmol/L, glucose at 16.7 mmol/L, andForskolin at 10 mM for 30 minutes, in a water bath at 37° C. Theperfusate is collected and insulin is assayed using a commerciallyavailable kit (e.g., Linco insulin kit). Insulin content of freeencapsulated islets and islets encapsulated with Sertoli cells isfurther examined via an acid-ethanol extract of said capsules andassayed for insulin content using a commercially available kit (e.g.Linco insulin kit).

In Vivo Encapsulation

Female Wistar-Furth rats are made diabetic by means of a single i.v.injection of streptozotocin. A total of 32 diabetic rats are dividedinto four groups and treated as follows: Group 1, a control group (n=8),will receive an intraperitoneal injection of at least 10 capsulescontaining Sertoli cells alone; Group 2 (n=8) will receive anintraperitoneal injection of 10 islets/g of free, non-encapsulatedislets; Group 3 (n=8) will receive an i.p. injection of 10 islets/g ofencapsulated islets alone; Group 4, (n=8) will receive 10 islets/g ofco-encapsulated islets plus Sertoli cells. No group will receive anyimmunosuppression following transplantation. All rats are closelymonitored via daily plasma glucose levels for the first weekpost-transplantation, and then at weekly intervals thereafter.

Rats are considered cured of diabetes if they exhibit a blood glucoselevel less than or equal to 170 mg/dl with concomitant steady increasesin body weight.

EXAMPLE 11

This example describes a method of immortalizing Sertoli cells using atemperature-sensitive mutant of the SV40 virus.

Sertoli cells are isolated from sexually mature (120 days) ratsaccording to the method of Wright et al. (1989) Ann. NY Acad. Sci.564:173-185. The testes are removed and the tubules are resuspended inDMEM F-12 (DMEM/F12: Life Technologies, Inc., Grand Island, N.Y.)containing 1 mg/ml collagenase (Worthington, Freehold, N.J.), 2mg/mlhyaluronidase (Sigma, St. Louis, Mo.), 0.3 mg/ml DNase (Sigma), and 65ug/ml soybean trypsin inhibitor (Sigma), and are incubated for 25 min at32° C. with gentle shaking. The incubation is repeated and the tubulesare washed in F12/DMEM and digested in an enzyme solution including 1mg/ml collagenase/dipase (Boehringer-Mannheim, Indianapolis, Ind.) inlieu of collagenase. The tubules are recovered and further broken up bygentle pipetting with a Pasteur pipette. The digestion mixture isfiltered through a nylon mesh to remove clumps of undispersed Sertolicells. The Sertoli cells are then sedimented at unit gravity yielding a95% pure population of adult Sertoli cells.

Two 25-cm² flasks are seeded with Sertoli cells at a density of 5×10⁶cells per plate. These flasks are incubated with SV40 virus mutanttsA255 for 3 h. The virus-containing medium is removed, and the cellsare incubated at 33° C. in F12/DMEM supplemented with 4% fetal bovineserum (FBS). Foci of transformed cells are visible at 6 weeks afterinfection. Each individual focus is isolated with sterile metal rings,the cells are isolated from the plate with trypsin EDTA, and the cellsuspensions are replated in 25-cm² flasks. Aliquots of cells from eachfocus are cultured at 33° C. or 40° C. for two days. At the end of thisculture period, the cells are collected and total RNA is isolated forNorthern blot analysis according to the methodology of Roberts, et al.(1992) Biol. Reprod. 47:92-96, incorporated herein by reference. Sertolicells are selected for cloning on the basis of the inducible expressionof mRNAs encoding Sertoli cell-secreted proteins according to themethods of Roberts, et al. (1995) Biol. Reprod. 53:1446-1453,incorporated herein by reference. Clonal cells are cultured in DMEM/F12Plus 4% FBS, supplemented with 1% antibiotic-antimycotic. The cells arethen seeded and allowed to attach at 33° C. for at least 24 hours.Clonal cells are collected by washing the plates twice with Hanksbalanced salt solution followed by a brief incubation with trypsin/EDTA.

What is claimed is:
 1. A method of treating a disease that results froma deficiency of a biological factor in a mammal wherein said methodcomprises administering Sertoli cells obtained from a cell line and atherapeutically effective amount of cells that produce said biologicalfactor to a mammal in need of such treatment, wherein said Sertoli cellsare administered in an amount effective to create an immunologicallyprivileged site.
 2. The method of claim 1 wherein said mammal is ahuman.
 3. The method of claim 1 wherein said biological factor is ahormone.
 4. The method of claim 1 wherein said biological factor isinsulin and said disease is diabetes mellitus.
 5. The method of claim 4wherein said cells that produce said biological factor are pancreaticislet of Langerhans cells.
 6. The method of claim 1 wherein said cellsthat produce said biological factor are cells transformed by a nucleicacid encoding said biological factor.
 7. The method of claim 1 whereinsaid administering is by transplantation.
 8. The method of claim 1wherein said Sertoli cells are administered in a dosage ranging from 10⁵to 10¹⁰ cells.
 9. The method of claim 1 wherein said cells that producesaid biological factor are administered in a dosage of from 10⁵ to 10¹⁰cells.
 10. The method of claim 7 wherein said transplantation is byxenograft.
 11. The method of claim 7 wherein said transplantation is byallograft.
 12. The method of claim 1 which further comprisesadministering an immunosuppressive agent.
 13. The method of claim 12wherein said immunosuppressive agent is administered for a timesufficient to permit said transplanted cells to be functional.
 14. Themethod of claim 12 wherein said immunosuppressive agent is cyclosporine.15. The method of claim 14 wherein said cyclosporine is administered ata dosage of from 5 to 40 mg/kg body wt.
 16. The method of claim 1 whichfurther comprises administering a therapeutically effective amount ofexogenous biological factor following the transplantation of said cellsthat produce said biological factor.
 17. The method of claim 1 whereinsaid cells that produce said biological factor are co-cultured withSertoli cells in tissue culture.
 18. The method of claim 17 wherein saidcells that produce said biological factor are cryopreserved prior toco-culturing with Sertoli cells in tissue culture.
 19. The method ofclaim 1 wherein said Sertoli cells are obtained by the stepscomprising:(a) isolating mammalian Sertoli cells from mammalian tissue;(b) incubating said isolated mammalian Sertoli cells with virusproducing cells under conditions sufficient to transform said Sertolicells; and (c) isolating said transformed cells from the virus producingcells.
 20. The method of claim 1 wherein said Sertoli cells are obtainedby the steps comprising:(a) isolating mammalian Sertoli cells frommammalian tissue; (b) incubating said isolated mammalian Sertoli cellswith a mutagen under conditions sufficient to transform said Sertolicells; and (c) collecting said transformed Sertoli cells.
 21. The methodof claim 19, wherein said virus producing cells are SV40 or polyomavirus.
 22. A method of treating diabetes mellitus in a mammal whereinsaid method comprises administering to a diabetic mammal Sertoli cellsobtained from a cell line in an amount effective to create animmunologically privileged site and a therapeutically effective amountof pancreatic islet of Langerhans cells.
 23. The method of claim 22wherein said diabetes mellitus is type I or type II.
 24. The method ofclaim 22 wherein said mammal is a human.
 25. The method of claim 22wherein said Sertoli cells are human, bovine or porcine.
 26. The methodof claim 22 wherein said pancreatic islet of Langerhans cells are human,bovine or porcine.
 27. The method of claim 22 wherein said administeringis by transplantation.
 28. The method of claim 27 wherein saidtransplantation is by injection into the renal subcapsular space. 29.The method of claim 27 wherein said transplantation is by injection intothe subcutaneous facie.
 30. The method of claim 22 wherein said Sertolicells are administered at a dosage ranging from 10⁵ to 10¹⁰ cells. 31.The method of claim 22 wherein said islet of Langerhans cells areadministered at a dosage ranging from 5-1000 islet cells/g body wt. 32.The method of claim 22 which further comprises the administration of animmunosuppressive agent.
 33. The method of claim 32 wherein saidimmunosuppressive agent is administered for a time sufficient to permitthe transplanted islets to be functional.
 34. The method of claim 32wherein said immunosuppressive agent is cyclosporine.
 35. The method ofclaim 32 wherein said cyclosporine is administered at a dosage of 5 to40 mg/kg body wt.
 36. The method of claim 22 which further comprisesadministering a therapeutically effective amount of insulin followingtransplantation of said pancreatic islet of Langerhans cells.
 37. Themethod of claim 22 wherein said Sertoli cells are obtained by the stepscomprising:(a) isolating mammalian Sertoli cells from mammalian tissue;(b) incubating said isolated mammalian Sertoli cells with virusproducing cells under conditions sufficient to transform said Sertolicells; and (c) isolating said transformed Sertoli cells from the virusproducing cell.
 38. The method of claim 22 wherein said Sertoli cellsare obtained by the steps comprising:(a) isolating mammalian Sertolicells from mammalian tissue; (b) incubating said isolated mammalianSertoli cells with a mutagen under conditions sufficient to transformsaid Sertoli cells; and (c) collecting said transformed Sertoli cells.39. The method of claim 37 wherein said virus producing cells are SV40or polyoma virus.
 40. A method of treating an autoimmune disease in amammal wherein said method comprises transplanting in to said mammal atherapeutically effective amount of isolated Sertoli cells obtained froma cell line to a transplant site in said mammal having said autoimmunedisease, wherein said site is other than testes.
 41. The method of claim40 wherein said Sertoli cells are administered in a dosage ranging from10⁵ to 10¹⁰ cells.
 42. The method of claim 40 wherein said Sertoli cellsare obtained by the steps comprising:(a) isolating mammalian Sertolicells from mammalian tissue; (b) incubating said isolated mammalianSertoli cells with virus producing cells under conditions sufficient totransform said Sertoli cells; and (c) collecting said transformedSertoli cells from the virus producing cell.
 43. The method of claim 40wherein said Sertoli cells are obtained by the steps comprising:(a)isolating mammalian Sertoli cells from mammalian tissue; (b) incubatingsaid isolated mammalian Sertoli cells with a mutagen under conditionssufficient to transform said Sertoli cells; and (c) isolating saidtransformed Sertoli cells.
 44. The method of claim 42 wherein said virusproducing cells are SV40 or polyoma virus.
 45. A method of enhancing therecovery and proliferation of ex vivo cells comprising co-culturing saidcells with Sertoli cells for a time and under conditions sufficient toachieve said enhanced recovery and proliferation.
 46. A method ofenhancing the recovery and proliferation of ex vivo cells comprisingculturing said cells in a tissue culture medium containing Sertoli cellsfor a time and under conditions sufficient to achieve said enhancedrecovery and proliferation.
 47. A method of treating a disease thatresults from a deficiency of a biological factor in a mammal whereinsaid method comprises administering Sertoli cells and a therapeuticallyeffective amount of cells that produce said biological factor to amammal in need of such treatment, wherein said Sertoli cells areadministered in an amount effective to create an immunologicallyprivileged site; and wherein said Sertoli cells and said cells thatproduce a biological factor are co-localized.
 48. The method of claim 47wherein said biological factor producing cells are co-localized byco-encapsulation.
 49. The method of claim 47 or 48 wherein said diseaseis diabetes mellitus and said biological factor producing cells areislet of Langerhans cells.
 50. The method of claim 19, 20, 37, 38, 42 or43 wherein said transformed Sertoli cells are screened for expression ofan appropriate isolate for cloning.